Brandon:LabNotes/Project1/2016-4-12

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scTHS-seq combinatorial indexing R5, human H1 P39 endoderm day 4 cells (differentiated)/mouse heart nuclei (scT7_r0XX_i5) and Tn5059[edit]

  • Performing THS-seq on H1 P39 endoderm day 4 differentiated cells and mouse heart nuclei. will use 1000-2000 cells per reaction, and less are viable. Mouse forebrain tissues from bings lab were grinded up, heart nuclei are not so maybe it will give better results.



Experiment/Protocol information[edit]

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
Tn5059 5 2.5 18 4.5 2.25
Total 36 9 4.5
Total % Glycerol 43.75
incubate at RT, 30m
store -20C


1. info

  • resuspend 1 tablet roche protease inhibitor in 1.5 mL N-H2O. Next day add 8.5 mL, then take 920 uL and add to 80 uL 50X LB
Repeater combitips
100  uL combitip at 1   uL =   2.5 uL 
100  uL combitip at 7   uL =   8.5 uL
200  uL combitip at 10  uL =  12.0 uL
200  uL combitip at 12  uL =  14.0 uL
500  uL combitip at 15  uL =  20.0 uL
1000 uL combitip at 150 uL = 157.0 uL 
  • 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.
  • FACS buffer: 2X (PBS, 4 mM EDTA, 2% BSA) (2X = 240 mg BSA, 96 uL 500 mM EDTA, 11.6 mL PBS) (1X = 3 mL 2X, 3 ml NH20)


  • 2. Samples
samples
before FACS sorting
1-96. scT7_r0XX_i5, 5X tn5059, ~2,000 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[edit]

  • 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 ~700 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


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


ONLY FOR R5, KEEP ON ICE, ADD CELLS TO TN5, THEN D6/4X LB MIXTURE

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 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
    • remove 57 uL of flow through (leaves extra so don't remove beads). Remove all excess during wash steps.
    • remove leftover EtOH with 1-10 uL LTS from all wells
    • ELUTE IN 8 + 2 uL taq5X, VORTEX LIGHTLY plate to resuspend. 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.

  • elute samples in 9.0 uL of N-H2O


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, VORTEX TO RESUSPEND
  • 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 14 uL N-H2O

  • 150 uL DNA binding buffer


18. PCR addition of barcodes USING NEXTERA XT V2 modified i5 (scT7_S5XX) AND i7 (N7XX) SEQUENCES!!!!!

KAPA SYBR FAST qPCR mix until saturation, X35 cycles

18 uL 2X kapa sybr fast
2  uL scT7_S5XX index primer, 2 uL F.
2  uL nXTv2_i7XX index primer, 2 uL R, (i7XX_long or i7XX_orig) USE_ORIG
14 uL DNA template
_____________
36 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. Run each sample on 6% PAGE gel, normalize concentrations. Pool samples, then Gel Size selection.

  • gel size select from 220-1000 bp, follow gel size selection protocol



Results[edit]

  • FACS results
  • FACS worked though lower capture of nuclei events at ~12%, usually 20-25%.

File:Sample1 crop.jpg


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

File:ZhangLab 2 2016-04-13 16hr 20min-labeled.jpg File:ZhangLab 2 2016-04-13 16hr 21min-invert-labeled.jpg


  • PCR curves

File:2016-04-14 scTHS-seq R5 differentiated hES cells, mouse heart cells bc addition worked.png File:2016-04-14 scTHS-seq R5 differentiated hES cells, mouse heart cells bc addition.png



  • 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

4/19/2016 intensity rm_blnk ng/ul size select amount 5ng 24985.26074 20438.97061 2.5ng 17109.17051 12707.68038 A 1 A1 44890.94134 10332.89031 1.732084814 2.88669467 total 404.9595403 B 1 B1 42128.13123 7570.080196 0.838698966 5.961614602 dye 40.49595403 C 1 C1 45612.63136 11054.58033 1.965451432 2.543944826 445.4554944 D 1 D1 45556.63136 10998.58033 1.947343199 2.567600823 E 1 E1 45466.54136 10908.49033 1.918211579 2.606594629 8 wells 55.6819368 F 1 F1 46012.05137 11454.00034 2.094608403 2.387081038 G 1 G1 44697.66127 10139.61024 1.669585522 2.994755245 H 1 H1 45078.80134 10520.75031 1.792831468 2.78888456 A 2 A2 44235.82132 9677.770288 1.520244358 3.288944948 B 2 B2 44785.9413 10227.89028 1.698131867 2.944412089 C 2 C2 44196.97132 9638.920287 1.507681771 3.316349707 D 2 D2 44406.91132 9848.860294 1.575568243 3.173458225 E 2 E2 52699.81157 18141.76054 4.257171184 1.174488829 F 2 F2 43621.5413 9063.49027 1.321609977 3.783264417 G 2 G2 44355.30126 9797.250232 1.558879547 3.207431909 H 2 H2 47392.32141 12834.27038 2.540934307 1.967780114 A 3 A3 48303.96144 13745.91041 2.835723405 1.763218511 B 3 B3 43900.82128 9342.770249 1.411918312 3.54128136 C 3 C3 47580.07142 13022.02039 2.601645392 1.921860687 D 3 D3 47264.09141 12706.04038 2.499469687 2.00042434 E 3 E3 58488.82174 23930.77071 6.129112994 0.815778728 F 3 F3 50705.03151 16146.98048 3.612136526 1.38422232 G 3 G3 51575.55148 17017.50045 3.893628987 1.284149059 H 3 H3 51912.54155 17354.49052 4.002598531 1.249188486 A 4 A4 48512.66145 13954.61042 2.903208908 1.722232246 B 4 B4 44863.43131 10305.38028 1.723189135 2.901596754 C 4 C4 45200.52135 10642.47032 1.832191006 2.728973117 D 4 D4 48057.86143 13499.8104 2.756144188 1.814128601 E 4 E4 52129.92155 17571.87052 4.072890811 1.227629277 F 4 F4 44171.27132 9613.220286 1.499371386 3.33473084 G 4 G4 46038.45131 11480.40028 2.103145122 2.377391815 H 4 H4 44179.48132 9621.430287 1.502026182 3.328836781 A 5 A5 43914.65131 9356.600279 1.416390408 3.530100155 B 5 B5 37274.70108 2716.650051 -0.730712478 0 C 5 C5 38952.02116 4393.970131 -0.188332089 0 D 5 D5 43918.89131 9360.840279 1.41776146 3.526686357 E 5 E5 50743.39151 16185.34048 3.624540665 1.379485144 F 5 F5 44802.68134 10244.63031 1.703544945 2.93505611 G 5 G5 40747.39115 6189.340125 0.392221072 12.74791274 H 5 H5 44571.63133 10013.5803 1.628832316 3.069683693 A 6 A6 38258.72114 3700.67011 -0.41251848 0 B 6 B6 40036.29116 5478.240133 0.16227886 12 C 6 C6 43045.10128 8487.050253 1.135211588 4.404465257 D 6 D6 48240.85144 13682.80041 2.815316073 1.775999522 E 6 E6 45872.79137 11314.74034 2.049577108 2.439527637 F 6 F6 44865.42134 10307.37031 1.723832633 2.900513602 G 6 G6 42618.58121 8060.530181 0.997291506 5.013579247 H 6 H6 45410.35135 10852.30032 1.900041908 2.631520905 A 7 A7 38536.51115 3978.460119 -0.322691944 0 B 7 B7 42025.59122 7467.540193 0.805541498 6.207004871 C 7 C7 50102.36149 15544.31046 3.41725637 1.463162098 D 7 D7 52114.07155 17556.02052 4.067765535 1.229176057 E 7 E7 45102.49134 10544.44031 1.800491897 2.777018885 F 7 F7 46190.59138 11632.54035 2.15234133 2.323051614 G 7 G7 43823.19125 9265.140217 1.386815764 3.605381572 H 7 H7 42336.76126 7778.710232 0.906161844 5.517778127 A 8 A8 49013.27146 14455.22043 3.06508681 1.631275168 B 8 B8 40791.55119 6233.500156 0.406500717 12.30010132 C 8 C8 41331.12123 6773.070202 0.580976784 8.606195872 D 8 D8 51345.75153 16787.7005 3.819320579 1.309133365 E 8 E8 60592.86181 26034.81078 6.809478108 0.734270662 F 8 F8 45007.69134 10449.64031 1.769837246 2.825118531 G 8 G8 42172.4612 7614.410167 0.853033562 5.861434087 H 8 H8 41297.70123 6739.650201 0.570170049 8.769313658 A 9 A9 41828.05125 7270.000217 0.741664716 6.741590767 B 9 B9 40139.27117 5581.220137 0.195578607 12 C 9 C9 38993.76116 4435.710132 0.1 12 D 9 D9 45871.11137 11313.06034 2.049033861 2.440174413 E 9 E9 44938.76134 10380.71031 1.747547951 2.861151819 F 9 F9 42252.33126 7694.280229 0.878860449 5.689185361 G 9 G9 38806.2711 4248.220067 0.1 12 H 9 H9 45923.07137 11365.02034 2.065835714 2.420327989 A 10 A10 41027.87122 6469.820193 0.482917469 10.35373603 B 10 B10 37061.47107 2503.420045 0.1 12 C 10 C10 42222.01126 7663.960228 0.869056134 5.753368284 D 10 D10 45859.11137 11301.06034 2.045153525 2.444804235 E 10 E10 41972.46125 7414.410221 0.788361321 6.342269547 F 10 F10 40065.07119 5507.020164 0.171585208 12 G 10 G10 42797.35122 8239.300186 1.055098806 4.73889267 H 10 H10 43990.88131 9432.830281 1.44104024 3.469715737 A 11 A11 43160.33129 8602.280256 1.172472511 4.264492304 B 11 B11 40162.28117 5604.230137 0.20301915 12 C 11 C11 41455.58124 6897.530206 0.621222332 8.048648198 D 11 D11 56455.21168 21897.16065 5.471522217 0.91382248 E 11 E11 43359.59129 8801.540262 1.236905484 4.042346052 F 11 F11 39883.77119 5325.720159 0.112959804 12 G 11 G11 41197.12117 6639.070138 0.53764635 9.299793438 H 11 H11 40455.41121 5897.360176 0.297806058 12 A 12 A12 40433.75118 5875.700145 0.290802043 12 B 12 B12 43496.04127 8937.990237 1.281028124 3.903114932 C 12 C12 43950.98128 9392.93025 1.428138115 3.501061941 D 12 D12 42492.00124 7933.950207 0.956360442 5.228154342


  • gel images

File:ZhangLab 2 2016-04-18 15hr 52min-labeled.jpg File:ZhangLab 2 2016-04-18 15hr 53min-labeled.jpg File:ZhangLab 2 2016-04-18 15hr 54min-labeled.jpg File:ZhangLab 2 2016-04-18 15hr 56min-labeled.jpg File:ZhangLab 2 2016-04-18 17hr 10min-labeled.jpg File:ZhangLab 2 2016-04-18 17hr 11min-labeled.jpg File:ZhangLab 2 2016-04-18 17hr 12min-labeled.jpg File:ZhangLab 2 2016-04-18 17hr 13min-labeled.jpg File:ZhangLab 2 2016-04-18 17hr 14min-labeled.jpg


  • gel size selection

File:ZhangLab 2 2016-04-19 15hr 10min-labeled.jpg File:ZhangLab 2 2016-04-19 15hr 11min-labeled.jpg


  • gel size selection valiation

File:ZhangLab 2 2016-04-20 11hr 50min-labeled.jpg