Brandon:LabNotes/Project1/2015-12-16

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THS-seq combinatorial indexing R1, pooled 96K cells mouse(F123 ES cells)/human(GM12878) (scT7_r0XX_i5) and Tn5059


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, 1,000 cells, buffer D6, samples 1-96

after FACS sorting
1-92. 25 nuclei, 1% BSA FACS buffer, gHCl after sort, pooled 1-96 samples
93. 500 cell positive control Rep1
94. 500 cell positive control Rep2
95. NTC control
96. NTC control


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
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

b. count cells in mouse and human cells and dilute to 333 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 1056 uL 2X 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. pool 1-96 into tube with 2X FACS buffer (1% BSA), so is 1X final FACS buffer.

b. spin down at 500 x g for 5 minutes, resuspend in 300 uL 1X sort buffer

c. Add PI stain to each tube, 15 uL each. (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 TA AND D6, 10% 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, ~10-15% of events were nuclei. was probably dirty from mixing with mouse ES cells which were plated on gelatin and had to be brought up with accutase

File:Batch Analysis 2015 12 16.tiff


  • TBU after IVT, single cell combinatorial indexing
  • A12 - D12. 25 human GM12878 / mouse F123 cells, 10 uL (whole reaction for gel)
  • E12. 500 human GM12878 / mouse F123 cells, 1 uL for gel
  • G12-H12. NTC, FACS sheath buffer, whole reaction for gel


' intensity ng/ul total
4532.763797 5
2132.932458 2.5
A12. 25 cells 783.5846339 1.094330566 1.094330566
B12. 25 cells 1151.700704 1.477811755 1.477811755
C12. 25 cells 1807.649147 2.161139404 2.161139404
D12. 25 cells 1616.98589 1.962517887 1.962517887
E12. 500 cells 4174.430366 4.626709776 46.26709776
F12 500 cells 3242.147971 3.65551403 36.5551403
G12. NTC 390.1517508 0.684475843 0.684475843
H12. NTC 44.51531534 0.324412586 0.324412586


  • gel
  • whole reaction for NTC is 10 uL

File:ZhangLab 2 2015-12-17 19hr 23min-labeled.jpg File:ZhangLab 2 2015-12-17 19hr 25min-labeled.jpg



Test on six 25 cell samples and controls

  • worked well so will process the rest of the samples
  • PCR curves

File:2015-12-18 scTHS-seq RNA processing test libraries.bmp

12/23/2015 ' ' ' ' ' ' '
2499.615864 5
1487.083729 2.5 for 50 ng *2
25 cells A11 6607.170395 15.14178807 3.30211992 6.604239841
25 cells B11 11910.86456 28.2369136 1.77073177 3.54146354
25 cells C11 9360.151844 21.93905739 2.279040485 4.558080971
25 cells D11 24062.18975 58.23923342 0.858527784 1.717055568
25 cells E11 19480.27317 46.92621822 1.06550244 2.131004879
25 cells F11 4519.732545 9.987784116 5.006115413 10.01223083
500 cells E12 29587.9675 71.88269609 0.695577694 1.391155389
500 cells F12 33092.05225 80.53448259 0.620852067 1.241704135 sum 35.19693515
NTC G11 348.1816111 -0.312014744 1 2 dye 7.039387029
NTC H11 119.1824496 -0.877426827 1 2
#DIV/0!
#DIV/0!


File:ZhangLab 2 2015-12-21 11hr 29min-labeled.jpg File:ZhangLab 2 2015-12-21 11hr 30min-invert-labeled.jpg


Processing of the rest of the samples, RNA processing and PCR barcoding


  • the qPCR machine gave a "voltage error" after 8 cycles, so restarted the machine and ran another 7 cycles. the libraries look a little over amplified, and look like the 500 cell samples when run for 15 cycles, when they only needed ~9 cycles. However should be fine, will run mi-seq and test.
  • PCR curves

File:2015-12-22 scTHS-seq RNA processing libraries rest1.bmp File:2015-12-22 scTHS-seq RNA processing libraries rest2.bmp


12/23/2016 ' ' ' ' ' ' '
intensity ng/ul
4935.006301 5
1749.18236 2.5 size select amt dye total
A1 5682.233182 5.586368625 2.685107448 22.4 135.2313129
B1 3769.715259 4.085565411 3.671462451 27.04626257
C1 2195.742785 2.850427734 5.262368108
D1 4118.312926 4.359119187 3.44106214
E1 2199.033371 2.853009943 5.257605231
A3 7215.256742 6.789372611 2.209335215
B3 10046.63837 9.011232391 1.664589187
C3 10596.09843 9.442408178 1.588577799
D3 8162.056185 7.532351084 1.991410097
E3 8246.830999 7.598876114 1.973976122
F3 6387.908015 6.140130262 2.442944915
A2 5409.844933 5.372618388 2.791934755
B2 9281.993181 8.411195158 1.78333753
C2 13239.02703 11.5163839 1.302492183
D2 9393.517292 8.498711067 1.764973522
E2 9944.62984 8.931183606 1.679508637
A4 17050.01723 14.50696833 1.033985851
B4 10925.13647 9.700612994 1.546294034
C4 6187.382425 5.982772547 2.507198775
D4 4867.636245 4.947132941 3.03205921
E4 8330.122738 7.664237337 1.957141897
F4 11681.23458 10.29394311 1.457167564
A5 10801.60806 9.603677001 1.561901759
B5 11984.79584 10.53215563 1.42420987
C5 8062.811208 7.454470935 2.012215237
D5 15156.9111 13.02139806 1.151950039
E5 6747.921491 6.422642324 2.335487365
F5 22632.00551 18.88730164 0.794184383
A6 10050.21524 9.014039248 1.664070855
B6 8686.149373 7.943620819 1.888307655
C6 9057.964953 8.235394304 1.821406413
D6 13225.85546 11.50604782 1.303662234
E6 14710.69322 12.67123914 1.183783199
F6 12971.15925 11.30618099 1.326707932
A7 7431.701526 6.959222536 2.155413183
B7 7796.667885 7.245621256 2.070215855
C7 9782.658031 8.804080059 1.70375552
D7 9693.692898 8.734266774 1.717373695
E7 9005.73766 8.19441017 1.830516131
F7 3500.129061 3.874014018 3.871952949
A8 8856.770391 8.077511628 1.85700754
B8 14507.68891 12.51193631 1.198855208
C8 14470.49739 12.48275114 1.201658179
D8 10944.94159 9.716154598 1.543820639
E8 10033.13916 9.000639201 1.666548304
F8 7992.216314 7.399073262 2.027280913
A9 13946.39042 12.07147058 1.242599226
B9 8733.84401 7.981048058 1.879452409
C9 7726.95023 7.190911975 2.085966294
D9 10083.42655 9.040101042 1.659273489
E9 12962.49664 11.29938321 1.327506088
F9 10232.93155 9.157421559 1.638015669
A10 20984.12112 17.59416333 0.852555459
B10 17957.82029 15.21934531 0.98558773
C10 12757.44145 11.138471 1.346683939
D10 4397.735007 4.578389057 3.276261543
E10 17135.02547 14.57367654 1.02925298
F10 15165.6988 13.028294 1.151340306


  • gel images

File:ZhangLab 2 2015-12-23 11hr 56min-labeled.jpg File:ZhangLab 2 2015-12-23 11hr 59min-labeled.jpg File:ZhangLab 2 2015-12-23 12hr 01min-labeled.jpg File:ZhangLab 2 2015-12-23 12hr 02min-labeled.jpg File:ZhangLab 2 2015-12-23 13hr 05min-lableled.jpg File:ZhangLab 2 2015-12-23 13hr 06min-labeled.jpg File:ZhangLab 2 2015-12-23 13hr 07min-labeled.jpg File:ZhangLab 2 2015-12-23 13hr 08min-labeled.jpg


  • gel size selection

File:ZhangLab 2 2015-12-23 16hr 16min-labeled.jpg File:ZhangLab 2 2015-12-23 16hr 19min-labeled.jpg


  • get PCR curve images
  • after gel size selection of libraries

File:ZhangLab 2 2015-12-23 19hr 38min-labeled.jpg