title: GSM7163189: JQPD_1_C2_4R; Mus musculus; ATAC-seq
library_construction_protocol: ATAC-seq was performed following the previously describe Omni-ATAC protocol. Briefly, viably frozen cells were pelleted by centrifugation and cell counts and viability was checked using Countess cell counter and NucBlue stain. 200,000 cells were resuspended in 1 ml of ice cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 in water). Cells were centrifuged at 800 r.c.f. for 5 min in a pre-chilled (4 °C) fixed-angle centrifuge and the supernatant was carefully aspirated by pipetting. Cell pellets were resuspended in 50 ?l of ATAC-seq RSB containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin by pipetting gently and incubated on ice for 3 min. To stop lysis, 1 ml of ATAC-seq RSB containing 0.1% Tween-20 (without NP40 or digitonin) was added and tubes were inverted to mix. Nuclei were then centrifuged for 10 min at 800 r.c.f. in a pre-chilled (4 °C) fixed-angle centrifuge. Supernatant was removed and nuclei were resuspended in 50 ?l of transposition mix (25 ?l 2× TD buffer, 2.5 ?l transposase Tn5 (Illumina 20034197), 16.5 ?l PBS, 0.5 ?l 1% digitonin, 0.5 ?l 10% Tween-20, and 5 ?l water) by pipetting gently. Transposition reactions were incubated at 37 °C for 30 min in a thermomixer at 1,000 r.p.m. Reactions were cleaned up with Qiagen MinElute kit. Library prep was done with qPCR optimization as described previously (PMID: 25559105). Amplified libraries were purified with Qiagen Qiaquick PCR purification columns as per the kit instructions and were quantified using Qubit.