GSM5903297: SKNAS_Taze2_Input; Homo sapiens; ChIP-Seq
Histone ChIP-sequencing was performed on 2 x 107 cells per reaction. Cells were crosslinked for 10 minutes in 1% formaldehyde and quenched in 0.20 M glycine for 5 minutes. Cells were lysed with SDS buffer supplemented with protease/phosphatase inhibitor cocktail and cell nuclei were pelleted. Nuclei pellets were resuspended in IP buffer and sheared for 15 minutes on a E220 Covaris sonicator (5% duty cycle, Peak power 140W, Cycles/burst 200) at 4C. Input lysates were separated from IP lysates, and the IP lysates were incubated overnight at 4C with a previously prepared mixture of antibody loaded on protein A or G magnetic beads. For ChIP-sequencing, 5 uL of drosophilia spike-in chromatin (ActiveMotif #53083) was added to each IP. Magnetic beads were washed twice with low salt wash buffer, twice with high salt wash buffer, twice with LiCl wash buffer, and once with cold TE. Washed DNA was eluted from beads with ChIP elution buffer. Input and IP samples were reverse crosslinked in high salt at 65C overnight. Uncrosslinked DNA was purified with SPRI beads and eluted for downstream analysis. Real-time quantitative RT-PCR for ST8SIA1 was performed using SYBR green (Bio-Rad) in combination with primers listed in Supplemental Table 4 and represented as percentage of input DNA. A gene desert on Chromosome 12 (ActiveMotif) was included as a negative control for antibody enrichment. DNA for ChIP-sequencing was submitted to the DFCI genomics core facility for library preparation and sequencing. DNA libraries were prepared with automated Swift S2 ligation chemistry and at least 80M 50 bp reads were sequenced on a NovaSeq platform.