title: GSM3443100: A1023C13: H3K9me2_untreated-2; Homo sapiens; ChIP-Seq
library_construction_protocol: The pelleted cells were washed twice with ice-cold PBS1x by centrifuging at 500 x g for 5 min at 4°C.  For lysis of crosslinked chromatin, the pellet was resuspended in lysis buffer A (50 mM Tris HCl pH8, 10 mM EDTA, 1% SDS, 50 x protease inhibitor cocktail) at room temperature and incubated for 5 min on rotating wheel. Next, lysate was centrifuged at 300 x g for 10 min at 8°C to prevent SDS precipitation and the supernatant was discarded. For chromatin shearing, the pellet was sheared in buffer B (25 mM Tris HCl pH8, 3 mM EDTA, 0.1% SDS, 1% Triton X-100, 150 mM NaCl, 50 x protease inhibitor cocktail) to approximatively 200-600 base-pair average size using the Bioruptor Pico (Diagenode). After centrifugation at 20 000 x g, 4ºC for 15 min, the supernatant containing sheared chromatin was used for immunoprecipitation. 25 µL (10%) of sheared chromatin was used as input DNA to normalize sequencing data. As supplement control for normalization, we used spike-in chromatin Drosophila and spike-in antibody (Active motif). For chromatin immunoprecipitation (1 million cells per ChIP), it was carried out using sheared chromatin and antibody H3K9me2 complexed to Dynal Protein G magnetic beads (Thermo Fisher). Briefly, 6 µL of H3K9me2 antibody was mixed with 1 µg of spike-in antibody. 23 µL of magnetic beads were washed three times in ice-cold buffer C (20 mM Tris HCl pH8, 2 mM EDTA, 0.1% SDS, 1% Triton X-100, 150 mM NaCl, 50 x protease inhibitor cocktail) and incubated with the mix of H3K9me2 and spike-in antibody for 4h at room temperature on rotating wheel in buffer C (494 µL). After the quick-spin and the removal of supernatant, the beads were resuspended in 100 µL buffer C. Each 50 µL of the latter was incubated with 250 µL of sheared chromatin mixed previously with 50 ng of spike-in chromatin Drosophila (250 µL chromatin of interest: 2,5 µL of spike-in chromatin, v:v) at 4°C on rotating wheel, overnight (~ 16h).  After quick-spin, the supernatant was discarded and the beads were successively washed in buffer C, buffer D (20 mM Tris-HCl pH8, 2 mM EDTA, 0.1% SDS, 1% Triton X-100, 500 mM NaCl), buffer E (10 mM Tris-HCl pH8, 0.25 M LiCl, 0.5% NP-40, 0.5% Sodium Deoxycholate, 1 mM EDTA) and buffer F (10 mM Tris-HCl pH8, 1 mM EDTA, 50 mM NaCl).  Finally, input and immunoprecipitated chromatin samples were resuspended in solution containing TE buffer/ 1% SDS, decrosslinked by heating at 65°C overnight and subjected to both RNase A and Proteinase K treatments. Illumina compatible libraries were prepared from input and immunoprecipitated DNAs using the Illumina TruSeq ChIP library preparation kit according to the manufacturer's protocol. Briefly, 4 to 10ng of DNA were subjected to subsequent steps of end-repair, dA-tailing and ligation of TruSeq indexed Illumina adapters. After a final PCR amplification step, the 24 resulting barcoded libraries were equimolarly pooled in 2 groups quantified using a qPCR method (KAPA library quantification kit, Roche) before sequencing on the Illumina HiSeq2500 instrument. Each pool was loaded on 1 rapid flow cell (11pM) and sequenced using a single read mode (SR50). This sequencing configuration was set to reach an average of 25 million reads (50-base long) per sample.