Brandon:LabNotes/Project1/2016-1-26: Difference between revisions

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>Bsos
>Bsos
Line 297: Line 297:


*FACS results
*FACS results
 
[[File:Humanbrain fetalmouse nuclei crop.jpg|600px]]




Line 307: Line 307:
[[File:ZhangLab 2 2016-01-27 20hr 23min-labeled.jpg|600px]]
[[File:ZhangLab 2 2016-01-27 20hr 23min-labeled.jpg|600px]]
[[File:ZhangLab 2 2016-01-27 20hr 24min-invert-labeled.jpg|600px]]
[[File:ZhangLab 2 2016-01-27 20hr 24min-invert-labeled.jpg|600px]]






*PCR curves
*PCR curves
[[File:2016-02-02 scTHS-seq bc mouse fetal and brain nuclei.bmp|600px]]




*plate layout for i5/i7 barcodes
*plate layout for i5/i7 barcodes
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''N701'''
| align="center" style="background:#f0f0f0;"|'''N702'''
| align="center" style="background:#f0f0f0;"|'''N703'''
| align="center" style="background:#f0f0f0;"|'''N704'''
| align="center" style="background:#f0f0f0;"|'''N705'''
| align="center" style="background:#f0f0f0;"|'''N706'''
| align="center" style="background:#f0f0f0;"|'''N707'''
| align="center" style="background:#f0f0f0;"|'''N710'''
| align="center" style="background:#f0f0f0;"|'''N711'''
| align="center" style="background:#f0f0f0;"|'''N712'''
| align="center" style="background:#f0f0f0;"|'''N714'''
| align="center" style="background:#f0f0f0;"|'''N715'''
|-
| ||||1||2||3||4||5||6||7||8||9||10||11||12
|-
| scT7_S502||A||A1, scT7_S502, N701||A2, scT7_S502, N702||A3, scT7_S502, N703||A4, scT7_S502, N704||A5, scT7_S502, N705||A6, scT7_S502, N706||A7, scT7_S502, N707||A8, scT7_S502, N710||A9, scT7_S502, N711||A10, scT7_S502, N712||A11, scT7_S502, N714||A12, scT7_S502, N715
|-
| scT7_S503||B||B1, scT7_S503, N701||B2, scT7_S503, N702||B3, scT7_S503, N703||B4, scT7_S503, N704||B5, scT7_S503, N705||B6, scT7_S503, N706||B7, scT7_S503, N707||B8, scT7_S503, N710||B9, scT7_S503, N711||B10, scT7_S503, N712||B11, scT7_S503, N714||B12, scT7_S503, N715
|-
| scT7_S505||C||C1, scT7_S505, N701||C2, scT7_S505, N702||C3, scT7_S505, N703||C4, scT7_S505, N704||C5, scT7_S505, N705||C6, scT7_S505, N706||C7, scT7_S505, N707||C8, scT7_S505, N710||C9, scT7_S505, N711||C10, scT7_S505, N712||C11, scT7_S505, N714||C12, scT7_S505, N715
|-
| scT7_S506||D||D1, scT7_S506, N701||D2, scT7_S506, N702||D3, scT7_S506, N703||D4, scT7_S506, N704||D5, scT7_S506, N705||D6, scT7_S506, N706||D7, scT7_S506, N707||D8, scT7_S506, N710||D9, scT7_S506, N711||D10, scT7_S506, N712||D11, scT7_S506, N714||D12, scT7_S506, N715
|-
| scT7_S507||E||E1, scT7_S507, N701||E2, scT7_S507, N702||E3, scT7_S507, N703||E4, scT7_S507, N704||E5, scT7_S507, N705||E6, scT7_S507, N706||E7, scT7_S507, N707||E8, scT7_S507, N710||E9, scT7_S507, N711||E10, scT7_S507, N712||E11, scT7_S507, N714||E12, scT7_S507, N715
|-
| scT7_S508||F||F1, scT7_S508, N701||F2, scT7_S508, N702||F3, scT7_S508, N703||F4, scT7_S508, N704||F5, scT7_S508, N705||F6, scT7_S508, N706||F7, scT7_S508, N707||F8, scT7_S508, N710||F9, scT7_S508, N711||F10, scT7_S508, N712||F11, scT7_S508, N714||F12, scT7_S508, N715
|-
| scT7_S510||G||G1, scT7_S510, N701||G2, scT7_S510, N702||G3, scT7_S510, N703||G4, scT7_S510, N704||G5, scT7_S510, N705||G6, scT7_S510, N706||G7, scT7_S510, N707||G8, scT7_S510, N710||G9, scT7_S510, N711||G10, scT7_S510, N712||G11, scT7_S510, N714||G12, scT7_S510, N715
|-
| scT7_S511||H||H1, scT7_S511, N701||H2, scT7_S511, N702||H3, scT7_S511, N703||H4, scT7_S511, N704||H5, scT7_S511, N705||H6, scT7_S511, N706||H7, scT7_S511, N707||H8, scT7_S511, N710||H9, scT7_S511, N711||H10, scT7_S511, N712||H11, scT7_S511, N714||H12, scT7_S511, N715
|-
|
|}




*gel quant
*gel quant
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''2/3/2016'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
|-
| sample||intensity||ng/ul||||||||
|-
| ||5034.611755||5||||||||
|-
| ||3223.284417||2.5||size select amt||||||
|-
| 1A||2976.701668||2.159665671||6.945519484||||total||159.2922454
|-
| 1B||2808.581288||1.927625365||7.781595049||||dye||31.85844908
|-
| 1C||7263.07745||8.075735745||1.857415903||||||191.1506945
|-
| 1D||9014.936439||10.49365733||1.429434898||||||
|-
| 1E||8136.990243||9.281913079||1.616046161||||7 wells||27.30724207
|-
| 1F||22956.08017||29.73527015||0.504451445||||||
|-
| 1G||8181.891294||9.343885659||1.605327863||||||
|-
| 1H||11042.49588||13.2921016||1.128489719||||||
|-
| 2A||12637.28827||15.49323935||0.968164221||||||
|-
| 2B||5936.802117||6.24520613||2.401842259||||||
|-
| 2C||10597.96542||12.67855919||1.183099734||||||
|-
| 2D||5832.807621||6.101672581||2.458342332||||||
|-
| 2E||10450.49031||12.47501355||1.202403504||||||
|-
| 2F||6522.883393||7.054117452||2.126417671||||||
|-
| 2G||16852.24998||21.31074347||0.703870328||||||
|-
| 2H||8777.822484||10.16639186||1.475449717||||||
|-
| 3A||9678.826736||11.40996092||1.314640787||||||
|-
| 3B||5126.122895||5.126303979||2.92608477||||||
|-
| 3C||7218.85596||8.014701097||1.871560751||||||
|-
| 3D||4350.954811||4.056414418||3.697847028||||||
|-
| 3E||10193.6503||12.12052212||1.237570449||||||
|-
| 3F||13529.7268||16.72498596||0.896861739||||||
|-
| 3G||9784.772604||11.55618776||1.298005909||||||
|-
| 3H||1488.3456||0.10543169||10||||||
|-
| 4A||29704.25786||39.04912738||0.384131503||||||
|-
| 4B||6892.974821||7.564918868||1.982836863||||||
|-
| 4C||8365.481313||9.597277213||1.562943288||||||
|-
| 4D||11175.08608||13.47510303||1.113164031||||||
|-
| 4E||5168.700154||5.185069253||2.892921824||||||
|-
| 4F||3762.178671||3.243783637||4.624229504||||||
|-
| 4G||10134.39078||12.03873192||1.245978405||||||
|-
| 4H||9100.105827||10.6112084||1.413599604||||||
|-
| 5A||13386.08566||16.52673199||0.907620455||||||
|-
| 5B||7934.771541||9.002810156||1.66614643||||||
|-
| 5C||6291.424868||6.734657628||2.227284716||||||
|-
| 5D||17084.71923||21.63159831||0.693430036||||||
|-
| 5E||18771.14056||23.95920262||0.626064241||||||
|-
| 5F||9525.370716||11.19816041||1.339505726||||||
|-
| 5G||5541.402715||5.699474564||2.631821553||||||
|-
| 5H||12586.35149||15.42293623||0.972577451||||||
|-
| 6A||16161.97509||20.35802378||0.736810221||||||
|-
| 6B||9149.775189||10.67976222||1.404525652||||||
|-
| 6C||12311.76632||15.04395288||0.997078369||||||
|-
| 6D||8342.36727||9.565375134||1.568155957||||||
|-
| 6E||7652.580228||8.61332877||1.741486991||||||
|-
| 6F||11647.9564||14.12776021||1.061739425||||||
|-
| 6G||18686.92719||23.84297105||0.629116227||||||
|-
| 6H||8069.824685||9.189210954||1.632349075||||||
|-
| 7A||26949.38238||35.24683911||0.425570076||||||
|-
| 7B||10242.30576||12.18767654||1.230751403||||||
|-
| 7C||12507.36293||15.31391595||0.979501262||||||
|-
| 7D||8876.831328||10.3030442||1.455880389||||||
|-
| 7E||9935.320296||11.76397418||1.275079303||||||
|-
| 7F||3874.584035||3.398925893||4.413158884||||||
|-
| 7G||10647.41139||12.74680468||1.176765502||||||
|-
| 7H||23019.73291||29.82312387||0.502965419||||||
|-
| 8A||21079.31865||27.14495768||0.552588815||||||
|-
| 8B||6438.083971||6.937077008||2.162294001||||||
|-
| 8C||20296.29699||26.06422858||0.575501399||||||
|-
| 8D||15494.79642||19.43718157||0.771716823||||||
|-
| 8E||6482.500193||6.998380425||2.143353046||||||
|-
| 8F||8868.812109||10.29197605||1.457446066||||||
|-
| 8G||27307.32415||35.74087153||0.419687583||||||
|-
| 8H||4546.166802||4.325846656||3.467529294||||||
|-
| 9A||18730.5074||23.9031206||0.627533126||||||
|-
| 9B||10668.17664||12.77546494||1.174125566||||||
|-
| 9C||20844.30998||26.82059794||0.559271648||||||
|-
| 9D||5969.194008||6.289913525||2.384770465||||||
|-
| 9E||10539.65031||12.59807248||1.190658335||||||
|-
| 9F||6179.253354||6.579838132||2.279691339||||||
|-
| 9G||22914.50983||29.67789463||0.505426688||||||
|-
| 9H||4428.09791||4.162887579||3.603268096||||||
|-
| 10A||5798.132673||6.053814103||2.477776777||||||
|-
| 10B||11280.10177||13.62004604||1.101317863||||||
|-
| 10C||10455.08521||12.48135544||1.201792552||||||
|-
| 10D||13476.13033||16.65101193||0.90084615||||||
|-
| 10E||17736.73053||22.53150646||0.665734447||||||
|-
| 10F||13524.29294||16.71748613||0.897264091||||||
|-
| 10G||12936.8649||15.906716||0.942997914||||||
|-
| 10H||17261.08051||21.87501274||0.685713886||||||
|-
| 11A||17762.00592||22.56639165||0.664705294||||||
|-
| 11B||12185.3122||14.86942048||1.008781749||||||
|-
| 11C||9892.768167||11.70524359||1.281476962||||||
|-
| 11D||9726.367319||11.47557659||1.307123863||||||
|-
| 11E||4016.11012||3.594260665||4.173320023||||||
|-
| 11F||2814.2753||1.93548426||7.749998442||||||
|-
| 11G||14956.11682||18.6936942||0.802409617||||||
|-
| 11H||16917.34939||21.4005939||0.700915127||||||
|-
| 12A||25644.98537||33.44650603||0.448477338||||||
|-
| 12B||24160.48722||31.39759675||0.477743571||||||
|-
| 12C||18501.80035||23.58745837||0.63593117||||||
|-
| 12D||10883.5706||13.07275239||1.147424777||||||
|-
|
|}




Line 343: Line 586:


*after gel size selection of libraries
*after gel size selection of libraries
[[File:ZhangLab 2 2016-02-04 11hr 53min-labeled.jpg|600px]]

Revision as of 20:08, 4 February 2016

scTHS-seq combinatorial indexing R2, human brain nuclei/mouse fetal tissue nuclei (scT7_r0XX_i5) and Tn5059

  • Performing THS-seq on human brain nuclei/mouse fetal tissue nuclei. will use ~3,000 nuclei per reaction since lose nuclei because they are sticky, and less are viable.



for 5X transposome ' ' ' ' '
Additional Control
Reagent Stock Conc (uM) Final rxn conc (uM) Vol (uL)
Glycerol 75% 75% 0.3 14.4 3.6 1.8
Annealed Transposon 50 5 3.6 0.9 0.45
EZTn5 5 2.5 18 4.5 2.25
Total 36 9 4.5
Total % Glycerol 43.75
incubate at RT, 30m
store -20C


Before starting protocols

1. Check if have enough reagents etc for the protocol

  • lysis buffer
  • tn5059 transposomes
  • custom 3' transpson
  • IVT reagents
  • cells etc
  • 5X, 2X taq polymerase
  • Zymo DNA clean and concentrator


  • 2. Samples
samples
before FACS sorting
1-96. scT7_r0XX_i5, 5X tn5059, 2,500 nuclei per rxn, buffer D6, samples 1-96

after FACS sorting
1-92. 25 nuclei, 1% BSA FACS buffer, gHCl after sort, pooled 1-96 samples
93. 25 nuclei positive IVT control 12E
94. 25 nuclei positive IVT control 12F
95. NTC control 12G
96. NTC control 12H


IVT Protocol

  • If need to make more transposome, do first 2 steps. If not goto step 3.

Generation of transposomes

1. annealing of ME sequence to T7 transposon sequence

a. Make 100 uM stock solution of tranposon and ME only bottom end.
b. Incubate 10 uL of each oligo (100uM) with at 95C for 2 minutes, then cool to 14C at 0.1 C/s.
c. Oligo's now at 50 uM in 20 uL.


2. Transposome complex generation, run controls!!!

  • add the below components into one tube and incubate for 30 minutes at RT
a. Add 3.6 uL of 75% sterile glycerol to tube
b. Add 0.9 uL of annealed transposon (50 uM) to the 75% sterile glycerol and mix well
c. Add 4.5 uL of transposase to well.
  • store at -20, is good for a year


3. Prepare samples, lyse cells with lysis buffer

CELL WASHING - (wash cells with PBS)

a. count cells, spin down all cells at 250Xg for 5 minutes SPIN DOWN NUCLEI 500X G
b. wash with 1X PBS, centrifuge 250Xg for 5 minutes
c. Count cells again to be sure of cells/uL number


CELL LYSIS AND MIXING

a. dilute Lysis Buffer (LB) to 4X add protease inhibitors, or as needed (from 10X stock)
80 uL 50X LB, 920 uL protease inhbiitors  KEEP NUCLEI/CELLS ON ICE

b. count cells in mouse and human cells and dilute to 833 cells/uL each.
   Mix human and mouse cells in equal volumes when at same concentration.
   add 3 uL cells to each well in 96 well plate.

c. add 1 uL 4X LB + protease inhibitors to each well, mix briefly

d. transposition ready


LYSIS BUFFER NOTES

  • Lysis buffer is: 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O.


3.5. start coating pooling tubes in 2056 uL 1X sort buffer. coat for 1 hour


4. transposition reaction. Add all components and incubate at 37C FOR 30 MINUTES

Added in 3 uL cells, then 1 uL 4X LB

4.0 uL lysed cells/pure genomic DNA
1.2 uL 5X Custom Tagmentation buffer USED BUFFER D6
1.2 uL T7 transposomes (5X tn5-059, indexed Tsome as listed)
___________
6.4 uL total solution

Incubate at 37C FOR 30 minutes


5. Stop reaction by adding (4.0 uL) 50 mM EDTA. (20 mM final EDTA) Incubate for 15 mins at 37C.


6. Nuclei pooling and recovery POOL ALL 96 SAMPLES

a. Add 1 volume (10 uL) 2X FACS buffer (PBS, 4 mM EDTA, 2% BSA) to each well, so is 1X final FACS buffer.

b. pool all samples into 1 tube and spin down at 500 x g for 5 minutes. resuspend in 400 uL 1X FACS buffer

c. Add PI stain to each tube, 20 uL. (5 uL per 100 uL cells).

d. take to FACS core for sorting. 25 nuclei per well, with some 500 nuclei (+) and NTC (-) controls
   -add 10 uL PBS to each well of sorting plate before hand
   -core checklist
   -put samples on ice to and from core.


add guanidine HCl when get back to lab

7. Add 11 uL 8M guanidine HCl for a total of 22 uL.

    • For SPRI beads add 1.8X (39 uL) beads per well and follow beads purification protocol
    • ELUTE IN 8, leave beads in solution.


8. Fill in reaction

  • Add 2 uL of 5X taq polymerase, mix thoroughly. Run at 72C for 3 minutes.


9. HiScribe T7 (NEB) T7 Protocol, IVT

added hiscribe mastermix directly into tubes after taq5x incubation

HiScribe (NEB) T7 protocol, IVT

a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) 
   on ice, but keep 10X transcription buffer at room temp

b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!!
 (10)     uL   DNA template (list 1 ug)
  2   uL   10X Transcription Buffer
  2   uL   100 mM ATP
  2   uL   100 mM CTP
  2   uL   100 mM GTP
  2   uL   100 mM UTP
  2   uL   T7 Enzyme Mix
 _______________________
  22 uL total 

c. Incubate reactions at 37C overnight for ~16-19 hours.

10. Clean with RNA Clean & Concentrator 5, put solution and all beads on columns.

  • using beads could be an option?
  • elute samples in 9.0 uL of N-H2O
  • quanitate/view on TBU gel, only 1 sample?


AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO


11. single strand synthesis MMLV RT (Clontech)

a. Add 2.5 uL 20 uM (100 ng total) RANDOM HEXAMERS to RNA sample. Bring to final volume of 11.5 uL with
   Nuclease free H2O

b. heat the mixture to 70C for 3 minutes. Immediately cool on ice.

c. Add the following to the reaction.
   4  uL 5X first strand buffer
   2  uL dNTP mix
   2  uL 100 mM DTT
  .5 uL SMART MMLV RT and mix (ADD LAST!!!!!)
  ___________________________
   20 uL total

d. Incuvate first at Room Temperature for 10 minutes. Then incubate at 42C for 60 minutes.

e. Terminate the reaction by heating at 70C for 10 minutes


12. RNase H digestion

  • Use .5 Units for 20 uL reaction. Stock is at 5 Units/ul, thus dilute 10X and use 1 uL per reaction.

a. Add 1 uL of 10X diluted Rnase H to the reaction.

b. Incubate for 20 minutes at 37C.


13. Second strand synthesis: Adding sss_scnXTv2 primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since sss_scnXTv2 has a high Tm.

a. Add 2.5 uL of 20 uM sss_scnXTv2 to each reaction.

b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice.

c. Add 5.875 uL taq5X to reactions, Incubate at 72C for 8 minutes.


14. Clean DNA with AMPURE beads depending.

  • USED AMPURE DNA BEADS AT 1:1.8X RATIO
  • add 54 uL beads per reaction
  • ADDED 7 uL N-H20 TO BEADS
  • LEFT BEADS IN SOLUTION, DID NOT REMOVE BEADS FROM ADDED N-H20


15. Fragmenting and 3' End tagging with [1X] custom nXTv2_i7 Tn5059 transposome

RAN TAGMENTATION ON BEADS

2 uL custom tagmentation buffer USED BUFFER D6, 16% FINAL DMF
X uL cDNA sample
X uL Nuclase free H2O
2 uL prepared 1X T7 transposomes (MAKE SURE TO ADD LAST)
___________
11 uL total solution

Incubate at 55C for 6 minutes, cool briefly on ice after


16. Add 4 uL N-H2O, then added 15 uL 8M guanidine HCl


17. Purified on DNA clean and concentrator, 5 volumes DNA binding buffer, elute in 13 uL N-H2O


18. PCR addition of barcodes USING NEXTERA XT V2 modified i5 (T7-1_S5XX) AND i7 (N7XX) SEQUENCES!!!!!

Samples:
1. jkd
2. dlfk
KAPA SYBR FAST qPCR mix until saturation, X35 cycles

17 uL 2X kapa sybr fast
2  uL scT7_S5XX index primer, 2 uL F, (i5XX_T7-1_long or short) USED XXX
2  uL nXTv2_i7XX index primer, 2 uL R, (i7XX_long or i7XX_orig)
13 uL DNA template (use half RT reaction)
_____________
34 uL

KAPA SYBR cycles:
72C 3m, 95C 30s, (95C for 10s, 63C for 30s, 72C for 1 min) X15, 72C for 3 min, 4C forever

  • terminate before curves saturate (usually cycle 6-8)


19. Pool all samples run on 6% PAGE gel, Qubit. Gel Size selection

  • gel size select from 220-1000 bp, follow gel size selection protocol
  • do not need to include controls.



Results

  • FACS results

File:Humanbrain fetalmouse nuclei crop.jpg




  • TBU after IVT, single cell combinatorial indexing. whole reactions used (10 uL)

File:ZhangLab 2 2016-01-27 20hr 23min-labeled.jpg File:ZhangLab 2 2016-01-27 20hr 24min-invert-labeled.jpg



  • PCR curves

File:2016-02-02 scTHS-seq bc mouse fetal and brain nuclei.bmp



  • plate layout for i5/i7 barcodes
' ' N701 N702 N703 N704 N705 N706 N707 N710 N711 N712 N714 N715
1 2 3 4 5 6 7 8 9 10 11 12
scT7_S502 A A1, scT7_S502, N701 A2, scT7_S502, N702 A3, scT7_S502, N703 A4, scT7_S502, N704 A5, scT7_S502, N705 A6, scT7_S502, N706 A7, scT7_S502, N707 A8, scT7_S502, N710 A9, scT7_S502, N711 A10, scT7_S502, N712 A11, scT7_S502, N714 A12, scT7_S502, N715
scT7_S503 B B1, scT7_S503, N701 B2, scT7_S503, N702 B3, scT7_S503, N703 B4, scT7_S503, N704 B5, scT7_S503, N705 B6, scT7_S503, N706 B7, scT7_S503, N707 B8, scT7_S503, N710 B9, scT7_S503, N711 B10, scT7_S503, N712 B11, scT7_S503, N714 B12, scT7_S503, N715
scT7_S505 C C1, scT7_S505, N701 C2, scT7_S505, N702 C3, scT7_S505, N703 C4, scT7_S505, N704 C5, scT7_S505, N705 C6, scT7_S505, N706 C7, scT7_S505, N707 C8, scT7_S505, N710 C9, scT7_S505, N711 C10, scT7_S505, N712 C11, scT7_S505, N714 C12, scT7_S505, N715
scT7_S506 D D1, scT7_S506, N701 D2, scT7_S506, N702 D3, scT7_S506, N703 D4, scT7_S506, N704 D5, scT7_S506, N705 D6, scT7_S506, N706 D7, scT7_S506, N707 D8, scT7_S506, N710 D9, scT7_S506, N711 D10, scT7_S506, N712 D11, scT7_S506, N714 D12, scT7_S506, N715
scT7_S507 E E1, scT7_S507, N701 E2, scT7_S507, N702 E3, scT7_S507, N703 E4, scT7_S507, N704 E5, scT7_S507, N705 E6, scT7_S507, N706 E7, scT7_S507, N707 E8, scT7_S507, N710 E9, scT7_S507, N711 E10, scT7_S507, N712 E11, scT7_S507, N714 E12, scT7_S507, N715
scT7_S508 F F1, scT7_S508, N701 F2, scT7_S508, N702 F3, scT7_S508, N703 F4, scT7_S508, N704 F5, scT7_S508, N705 F6, scT7_S508, N706 F7, scT7_S508, N707 F8, scT7_S508, N710 F9, scT7_S508, N711 F10, scT7_S508, N712 F11, scT7_S508, N714 F12, scT7_S508, N715
scT7_S510 G G1, scT7_S510, N701 G2, scT7_S510, N702 G3, scT7_S510, N703 G4, scT7_S510, N704 G5, scT7_S510, N705 G6, scT7_S510, N706 G7, scT7_S510, N707 G8, scT7_S510, N710 G9, scT7_S510, N711 G10, scT7_S510, N712 G11, scT7_S510, N714 G12, scT7_S510, N715
scT7_S511 H H1, scT7_S511, N701 H2, scT7_S511, N702 H3, scT7_S511, N703 H4, scT7_S511, N704 H5, scT7_S511, N705 H6, scT7_S511, N706 H7, scT7_S511, N707 H8, scT7_S511, N710 H9, scT7_S511, N711 H10, scT7_S511, N712 H11, scT7_S511, N714 H12, scT7_S511, N715



  • gel quant
2/3/2016 ' ' ' ' ' '
sample intensity ng/ul
5034.611755 5
3223.284417 2.5 size select amt
1A 2976.701668 2.159665671 6.945519484 total 159.2922454
1B 2808.581288 1.927625365 7.781595049 dye 31.85844908
1C 7263.07745 8.075735745 1.857415903 191.1506945
1D 9014.936439 10.49365733 1.429434898
1E 8136.990243 9.281913079 1.616046161 7 wells 27.30724207
1F 22956.08017 29.73527015 0.504451445
1G 8181.891294 9.343885659 1.605327863
1H 11042.49588 13.2921016 1.128489719
2A 12637.28827 15.49323935 0.968164221
2B 5936.802117 6.24520613 2.401842259
2C 10597.96542 12.67855919 1.183099734
2D 5832.807621 6.101672581 2.458342332
2E 10450.49031 12.47501355 1.202403504
2F 6522.883393 7.054117452 2.126417671
2G 16852.24998 21.31074347 0.703870328
2H 8777.822484 10.16639186 1.475449717
3A 9678.826736 11.40996092 1.314640787
3B 5126.122895 5.126303979 2.92608477
3C 7218.85596 8.014701097 1.871560751
3D 4350.954811 4.056414418 3.697847028
3E 10193.6503 12.12052212 1.237570449
3F 13529.7268 16.72498596 0.896861739
3G 9784.772604 11.55618776 1.298005909
3H 1488.3456 0.10543169 10
4A 29704.25786 39.04912738 0.384131503
4B 6892.974821 7.564918868 1.982836863
4C 8365.481313 9.597277213 1.562943288
4D 11175.08608 13.47510303 1.113164031
4E 5168.700154 5.185069253 2.892921824
4F 3762.178671 3.243783637 4.624229504
4G 10134.39078 12.03873192 1.245978405
4H 9100.105827 10.6112084 1.413599604
5A 13386.08566 16.52673199 0.907620455
5B 7934.771541 9.002810156 1.66614643
5C 6291.424868 6.734657628 2.227284716
5D 17084.71923 21.63159831 0.693430036
5E 18771.14056 23.95920262 0.626064241
5F 9525.370716 11.19816041 1.339505726
5G 5541.402715 5.699474564 2.631821553
5H 12586.35149 15.42293623 0.972577451
6A 16161.97509 20.35802378 0.736810221
6B 9149.775189 10.67976222 1.404525652
6C 12311.76632 15.04395288 0.997078369
6D 8342.36727 9.565375134 1.568155957
6E 7652.580228 8.61332877 1.741486991
6F 11647.9564 14.12776021 1.061739425
6G 18686.92719 23.84297105 0.629116227
6H 8069.824685 9.189210954 1.632349075
7A 26949.38238 35.24683911 0.425570076
7B 10242.30576 12.18767654 1.230751403
7C 12507.36293 15.31391595 0.979501262
7D 8876.831328 10.3030442 1.455880389
7E 9935.320296 11.76397418 1.275079303
7F 3874.584035 3.398925893 4.413158884
7G 10647.41139 12.74680468 1.176765502
7H 23019.73291 29.82312387 0.502965419
8A 21079.31865 27.14495768 0.552588815
8B 6438.083971 6.937077008 2.162294001
8C 20296.29699 26.06422858 0.575501399
8D 15494.79642 19.43718157 0.771716823
8E 6482.500193 6.998380425 2.143353046
8F 8868.812109 10.29197605 1.457446066
8G 27307.32415 35.74087153 0.419687583
8H 4546.166802 4.325846656 3.467529294
9A 18730.5074 23.9031206 0.627533126
9B 10668.17664 12.77546494 1.174125566
9C 20844.30998 26.82059794 0.559271648
9D 5969.194008 6.289913525 2.384770465
9E 10539.65031 12.59807248 1.190658335
9F 6179.253354 6.579838132 2.279691339
9G 22914.50983 29.67789463 0.505426688
9H 4428.09791 4.162887579 3.603268096
10A 5798.132673 6.053814103 2.477776777
10B 11280.10177 13.62004604 1.101317863
10C 10455.08521 12.48135544 1.201792552
10D 13476.13033 16.65101193 0.90084615
10E 17736.73053 22.53150646 0.665734447
10F 13524.29294 16.71748613 0.897264091
10G 12936.8649 15.906716 0.942997914
10H 17261.08051 21.87501274 0.685713886
11A 17762.00592 22.56639165 0.664705294
11B 12185.3122 14.86942048 1.008781749
11C 9892.768167 11.70524359 1.281476962
11D 9726.367319 11.47557659 1.307123863
11E 4016.11012 3.594260665 4.173320023
11F 2814.2753 1.93548426 7.749998442
11G 14956.11682 18.6936942 0.802409617
11H 16917.34939 21.4005939 0.700915127
12A 25644.98537 33.44650603 0.448477338
12B 24160.48722 31.39759675 0.477743571
12C 18501.80035 23.58745837 0.63593117
12D 10883.5706 13.07275239 1.147424777


  • gel images

File:ZhangLab 2 2016-02-03 11hr 53min-labeled.jpg File:ZhangLab 2 2016-02-03 11hr 56min-labeled.jpg File:ZhangLab 2 2016-02-03 11hr 58min-labeled.jpg File:ZhangLab 2 2016-02-03 11hr 59min-labeled.jpg File:ZhangLab 2 2016-02-03 15hr 21min-labeled.jpg File:ZhangLab 2 2016-02-03 15hr 23min-labeled.jpg File:ZhangLab 2 2016-02-03 15hr 24min-labeled.jpg File:ZhangLab 2 2016-02-03 15hr 26min-labeled.jpg File:ZhangLab 2 2016-02-03 15hr 27min-labeled.jpg




  • gel size selection

File:ZhangLab 2 2016-02-03 19hr 53min-labeled.jpg File:ZhangLab 2 2016-02-03 19hr 54min-labeled.jpg


  • after gel size selection of libraries

File:ZhangLab 2 2016-02-04 11hr 53min-labeled.jpg