GSM3523738: WS-sgNTC-H3K4me3-rep1; Homo sapiens; ChIP-Seq
One million cells per sample were cross-linked by 1% vol/vol formaldehyde for 8 or 12 minutes at room temperature, if for acetylated ChIP, add additional 20 mM sodium butyrate. Samples were then re-suspended in lysis buffer (50 mM Tris-HCl, 10 mM EDTA, 1% SDS, pH8.0) for 5 min on ice. Subsequently, lysates were sonicated using a Covaris S2 instrument. Cells were harvested and DNA was extracted 28, 42 days post-infection (day28, day42), as well as at the end of the experiment (day56) using the Qiagen DNeasy Blood and Tissue Kit to evaluate library representation at middle and final time points. And the initial point are represented by plasmid sequencing. PCR on gDNA was performed using PrimeSTAR HS DNA Polymerase following manufacturer's instructions (Takara). A two-step PCR method was used to amplify inserted sgRNA sequences and append molecular barcodes and Illumina adapter sequences. In the first PCR, primers were designed to target the flanking sgRNA sequences with a biotin in forward primer. Reaction conditions were as follows: 98 °C denature for 2 minutes followed by 14cycles of 98 °C for 10 seconds, 58.0 °C anneal for 30 seconds, 72 °C extension for 40 seconds, and a final single extension step at 72 °C for 5 minutes. Multiple reactions per library were performed to ensure adequate input library coverage, then combined and purified by C1 beads (Invitrogen) and used as a template for the second PCR. The second PCR primers added the Illumina Hi-Seq2500 adapter sequences and barcode sequences. The reaction conditions were the same as the first PCR except only 12 cycles were used.
GEO Accession
GSM3523738