Brandon:LabNotes/Project1/2015-12-16

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THS-seq combinatorial indexing R1, pooled 96K cells mouse/human (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 P-sss_nXTv2 primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since P_sss_nXTv2 has a high Tm.

a. Add 2.5 uL of 20 uM P-sss_nXTv2 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

  • blah