Alice:Whole Genome Bisulfite Sequencing Lab Notes/methylation reporter

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Revision as of 00:43, 13 January 2016 by >Zsakura2 (Created page with "=methylation reporter knock-in= *The methylation reporter is developed by Jaenisch group: [http://www.cell.com/cell/abstract/S0092-8674(15)01098-3 Link] *Tracing Dynamic Chang...")
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methylation reporter knock-in

  • 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 and cell culture protocol

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
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
Transfection using Lipofectamin 3000 kit:
1-1-2016: 
passaged four cell lines at 1:20 dilution (SCNT NB3, SCNT B12, miPS 1E12S5, miPS B3)
1-2-2016: 
transfection following manufacture protocol Link
Highlights:
combination of these two plasmids needed: cas9+sox2 (CRISPR and gRNA on one plasmid design), Sox2SE donor plasmid (ordered from addgene)
cas9+sox2 (611.37 ng/ul) 25 ul total for 4 reactions
Sox2 SE donor (1ug/ul)