title: GSM5233374: ATACseq_Setdb1KO_rep2; Mus musculus; OTHER
library_construction_protocol: 1.10^6 cells were used for each Omni-ATAC-seq experiment.  If there is 5 to 15% of death cells, cells are incubated with DNAse I at 37°C for 30min then cells are collected at 1200g for 3min at 4°C and washed 3 times with 1mL ice-cold PBS. Cells were resuspended into 1mL ice-cold PBS, 50,000 cells (50µL) were transfered into fresh 1,5mL Eppendorf tube. Omni-ATAC-seq was performed as described in Corces et al 2017, Nature Methods. Transposase enzyme was from Netera XT DNA library Preparation Kit. Omni-ATAC-seq library preparation was performed as described in Buesnrostro et al 2013 Nature Methods with few adaptations. Illumina sequencing primers was from Buenrostro et al 2013 Nature Methods. To determine the number of additional cycles required, Ct corresponding to plateau is identified and ¼ of Ct + 1 defined the number of additional cycles. Then Omni-ATAC-seq libraries were cleaned up with AMPure XP beads to remove DNA fragment smaller than 100bp and longer than 1,5Kb. 30µL H2O was added to libraries obtain a final volume of 50µL then 25µL AMPure XP beads were added and incubate at RT for 15min. AMPure XP beads are placed on magnetic stand for 5min at RT. Supernatants were transfered into fresh tube and 65µL AMPure XP beads were added to samples then incubate at RT for 15min and place on magnetic stand for 5min at RT. Supernatant was removed. AMPure XP beads were whased with 200µL fresh 80%-EtOH twice. AMPure XP beads were air-dry for 7min at RT. 22µL Buffer EB was added to resuspend AMPure XP beads then they are incubated at RT for 2min and placed on magnetic stand for 5min at RT. 20µL ATAC-library samples were transferred into fresh 1,5mL Eppendorf tube. 1µL of library was diluted 1:100 to do qPCR quality control and to run Agilent High Sensitivity DNA Electrophoresis to check DNA fragment size. Before sequencing, ATAC-seq libraries are quantified by qPCR. Libraries were sequenced 75bp paired-end with 50 million reads of depth sequencing on HiSeq 2000 Illumina machine at the MGX platforms.