GSM7889152: A549, H3K9me3, ChIP-ISO input, biol rep 1; Homo sapiens; OTHER
Cells were fixed with formaldehyde (1% final concentration) for 10 minutes at room temperature. Crosslinking was quenched with glycine (0.125M final concentration) for 5 minutes at room temperature. After cell and nuclei lysis, chromatin was fragmented in Diagenode Pico with a circulating water bath at 4°C using the Shear and Go Easy Mode setting for 3 cycles (30 seconds on followed by 30 seconds off). To amplify the integrated ChIP-ISO library sequences while excluding other genomic DNA, including native CCND1e, we performed two rounds of PCR amplification with a BbsI digestion step in between. The primer pairs for the first round of PCR contain regions annealing to the CCND1e (outside the 193bp library sequence) at the 3'-ends, partial Illumina TruSeq adaptor sequences at the 5'-end and 0-3 random nucleotide spacers in between to increase sequence complexity. Primers with different numbers of spacers are mixed in equimolar ratio for the first round of PCR. Preliminary PCR tests were performed to decide the optimal cycle number that keeps the PCR reactions in exponential phase. We used 23-25 cycles for our first round of PCR. For the first round of PCR, 30 μl of ChIP DNA was amplified in a 100 μl PCR reaction using NEBNext Ultra II Q5 master mix. The PCR products were purified with AMPure XP beads with a beads to DNA ratio of 0.9. 15 μL of the purified PCR product was digested with 20 U of BbsI in a 30 μL reaction at 37°C for two hours. The digestion products were purified with AMPure XP beads with a beads to DNA ratio of 0.9, followed by the second round of PCR. The primer pairs for the second round of PCR contain the rest of the Illumina TruSeq adaptor sequences and sample indexes. For the second round of PCR, 2 μl of purified digestion product was amplified in a 50 μl PCR reaction for 8 cycles using NEBNext Ultra II Q5 master mix. The PCR products were purified with AMPure XP beads with a beads to DNA ratio of 0.8. Quality control was conducted with TapeStation (Agilent). 30 million paired-end 150bp reads were obtained for each ChIP-ISO and input sample using a NextSeq 2000 (Illumina) instrument.