Alice:Whole Genome Bisulfite Sequencing Lab Notes/methylation reporter: Difference between revisions

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  transfection following manufacture protocol [https://tools.thermofisher.com/content/sfs/manuals/lipofectamine3000_protocol.pdf Link]
  transfection following manufacture protocol [https://tools.thermofisher.com/content/sfs/manuals/lipofectamine3000_protocol.pdf Link]
  plasmids were added according to the table below
  plasmids were added according to the table below
NOTES: needs to calculate the ratio of cas9 and donor plasmid carefully based on 1:1 molar ratio


  1-4-2016:
  1-4-2016:
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| align="center" style="background:#f0f0f0;"|'''notes'''
| align="center" style="background:#f0f0f0;"|'''notes'''
|-
|-
| cas9+sox2||611.37||25||4||added together to cell line of interest with donor plasmid
| cas9+sox2||611.37||25||4||added to cell line of interest together with donor plasmid
|-
|-
| Sox2 SE donor||1000||20||4||added together to cell line of interest with cas9+gRNA plasmid
| Sox2 SE donor||1000||20||4||added to cell line of interest together with cas9+gRNA plasmid
|-
|-
| pLk0.1 GFP||429||20||2||control plasmid to test transfection efficiency
| pLk0.1 GFP||429||20||2||control plasmid to test transfection efficiency
|}
|}

Latest revision as of 18:29, 13 January 2016

methylation reporter knock-in[edit]

  • The methylation reporter is developed by Jaenisch group: Link
  • Tracing Dynamic Changes of DNA Methylation at Single-Cell Resolution. Stelzer Y, Shivalila CS, Soldner F, Markoulaki S, Jaenisch R. Cell. 2015 Sep 24;163(1):218-29. doi: 10.1016/j.cell.2015.08.046.
  • I would like to take advantage of this reporter to trace the methylation changes at my locus-of-interest

cloning protocol[edit]

Sox2se-CPG-TV plasmid maxi-prep:
purchased these two plasmids from addgene: SOX2se-CPG-TV (Plasmid #70155); GAPDH-CPG-TV (Plasmid #70148)
shake the plasmids in 3ml AmpR LB for 12 hours at 37C, then transfer to 300ml AmpR LB media and shake overnight at 37C 
use Qiagen maxi-prep kit to extract plasmid DNA from the overnight culture
take 100ug of the extracted DNA of Sox2SE sample, and perform overnight digestion with restriction enzyme BamHI

donor plasmid: Sox2SE
Insert sox2 gRNA into cas9-mcherry plasmid:
digest p2U6-pCAG-Cas9-mChery plasmid with BaeI
ligate Sox2SE oligo with T4 ligase
transform into TOP10 or Sblt3 chemical competent cells
plate overnight
pick single clone and shake in 3ml AmpR LB media overnight
Sanger sequencing to double check for correct insert 

Notes:
To use with p2U6-pCAG-Cas9-mcherry plasmid (cas9 and gRNA on one plasmid construct):
Forward: N1-N20-GTTTT
Reverse: N20-N1-CGGTG

Published sequences:
D1 SOX2-SE Crispr	CACCGCCAGCTTTCCGAGCCAGATG
D2 SOX2-SE Crispr	AAACCATCTGGCTCGGAAAGCTGGC

Oligos ordered:
SOX2-SE-F: CCAGCTTTCCGAGCCAGATGGTTTT
SOX2-SE-R: CATCTGGCTCGGAAAGCTGGCCGGTG

CRISPR and gRNA plasmid: cas9+sox2se 

Transfection[edit]

1-1-2016: 
passaged four cell lines at 1:20 dilution (SCNT NB3, SCNT B12, miPS 1E12S5, miPS B3)
1-2-2016: 
transfect the cells using lipofectamin 3000 kit
transfection following manufacture protocol Link
plasmids were added according to the table below
NOTES: needs to calculate the ratio of cas9 and donor plasmid carefully based on 1:1 molar ratio
1-4-2016:
added puro to selection out untransfected cells (Puro stock is 2mg/ml, and dilute to 0.5mg/ml for usage)
1-8-2016:
passaged the following cell lines at 1:1 ratio onto another 1 well of 6-well plate each
SCNT NB3 P12
SCNT B12 P15
miPS 1E12S5 P23
miPS B3 P12
1-11-2016:
FACS sorting at Sanford consortium core facility
sort 20,000 cells RFP positive and negative cells into 2 well of 24 well plate each (total of 16 wells for 4 samples)
plasmid concentration (ng/ul) amount (ul) total # of reactions notes
cas9+sox2 611.37 25 4 added to cell line of interest together with donor plasmid
Sox2 SE donor 1000 20 4 added to cell line of interest together with cas9+gRNA plasmid
pLk0.1 GFP 429 20 2 control plasmid to test transfection efficiency