GSM8960452: WT, Op9DL1_24h, ATAC-seq, rep1; Mus musculus; ATAC-seq
Sorted cells are spun down (500xg, 4 °C, 5 min) and washed in 50 µl cold ATAC-RSB (10 mM Tris-Hcl pH 7.4, 10 mM NaCl, 3 mM MgCl2), followed by 3 min incubation on ice in ATAC-RSB supplemented with 0.1% NP40, 0.1 % Tween-20 and 0.01 % digitonin. 1 ml ATAC-RSB containing 0.1 % Tween-20 only was added and the samples were spun down at 500xg, 4°C for 10 min. Supernatants were discarded and the nuclei were resuspended in transposition reaction mix (25 µl Tagment DNA buffer (Illumina, 15028212), 2.5 µl Tagment DNA enzyme (Illumna, 15028212), 16.5 µl PBS, 0.001 % Digitonin, 0.1 % Tween-20, and Nuclease free H20 waster up to 50µl) and the tagmentation reactions were run by incubating the samples at 37 °C for 30 min. Samples were purified using ChIP clean and Concentrator kit (Zymo Research, D5205) and eluted in 10 µl elution buffer. To each sample, 25 µl NEBNext Ultra II Q5 Master mix (New England Biolabs, M0544L), 3 µl 10x SYBR green (Invitrogen, S-7563), 6.25 µl 10 µM Nextera primer 1 (no barcode) as well as 6.25 µl of 10 µM Nextera primer 2 (barcode) unique for each sample. The transposed elements were amplified by running the following thermo-cycler program: 72 °C 5min, 98 °C 30 s, N x (98 °C 10 s, 63 °C 30 s, 72°C 30 s). N are defined as the number of cycles that correspond to ¼ of max intensity and was determined by running 5 µl of sample after 5 rounds of amplification on q-PCR. Amplified libraries were purified with Ampure XP (1.2 x sample volume) and quality control were performed with using Qubit® dsDNA HS Assay Kit & assay tubes (ThermoFisher Scientific, Q32854/Q32856) and High Sensitivity DNA Kit (Agilent, 5067-4626). Omni-ATAC-seq libraries were subjected to 2x38 cycles of paired-end sequencing on a Nextseq500 (Illumina) using the NextSeq 500/550 High Output Kit v2.5 (Illumina, 20024906).