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''' | ||
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| cas9+sox2||611.37||25||4||added | | cas9+sox2||611.37||25||4||added to cell line of interest together with donor plasmid | ||
|- | |- | ||
| Sox2 SE donor||1000||20||4||added | | 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 | ||
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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 |