GSM6595240: hsSKM_ChIP_H3_rep1; Homo sapiens; ChIP-Seq
The samples were extracted using a SimpleChIP Enzymatic Chromatin IP kit (#9003; Cell Signaling Technologies). Cells were cross-linked with 1 % formaldehyde in DMEM complete medium at room temperature for 10 min, neutralized with 10 % 1 M glycine at room temperature for 5 min and washed with PBS. Fixed cells were lysed with 1 ml Buffer A+DTT+PIC for 10 min on ice to isolate nuclear. Isolated nuclear were washed with buffer B+DTT for 3 times then diluted in 100 ul buffer B+DTT and Chromatin were digested with 0.5 ul Micrococcal Nuclease #10011 at 37°C for 20 mins, then 10 μl 0.5 M EDTA #7011 was added to terminate digestion. Digested nuclear were diluted in 500ul ChIP buffer, sonicated with ultrasonic processor (Ningbo sinentz biotechnology CO.,LTD) at sonications of 5 s with 30-s intervals, 12 times at 20 % AMPL, and cleared by centrifugation at 9400 g for 10 min at 4°C. The supernatants were incubated with 3 ug rabbit anti-UBE2A antibody (PA5-29940, invtrogen) or rabbit anti-histone H3 (a technical positive control, #4620, Cell Signaling Technologies) overnight with rotation, then incubated with 30 ul Protein G Magnetic Beads (#9006) for 2 hours with rotation.   The immune complexes were washed with 1 ml low salt buffer (1XChIP buffer) for 3 times, 1 ml high salt buffer (1XChIP buffer+350 mM NaCl) for 1 time, then eluted with 150 ul ChIP elution buffer at 65 °C for 30  min with rotation.   6 ul 5 M NaCl and 2 ul Proteinase K #10012 was added to the eluted DNA. The DNA were incubated at 65°C for 2 hours to reserve crosslink. Then the DNA fragments were purified according to the manufacturer's instructions. Libraries were prepared according to KAPA's instructions accompanying the DNA Sample Kit. Briefly, DNA was end-repaired using a combination of 3'-5' exonuclease and polymerase. A protruding 3- 'A' base is introduced for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with primers and library fragments of ~ 380 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina NovaSeq 6000 following the manufacturer's protocols.