GSM8031898: ATAC-seq on purified Müller cells from 6-month-old db/db mice - rep2; Mus musculus; ATAC-seq
Müller cells were purified using the MACS protocol and subjected to the omni-ATAC protocol with slight modification [PMID: 28846090]. Briefly, 100.000 viable, pelleted cells were resupended in cold ATAC resuspension buffer (10mM Tris-HCl pH 7.5 (Invitrogen, catalog nr 15567027), 10mM NaCl (Invitrogen, catalog nr AM9760G), 5mM MgCl2 (Invitrogen, catalog nr AM9530G), 0.1% IgePAL (Sigma Aldrich, catalog nr I3021-50ML), 0.1% Tween-20 (Sigma Aldrich, catalog nr P9416-50ML), 0.01% Digitonin (Promega, catalog nr G9441)) and incubated on ice for 3min. Lysed cells were washed with cold ATAC wash buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 5mM MgCl2, 0.1% Tween-20) and nuclei pelleted 10min at 500g. Nuclei were resuspended in 25uL of transposition mix (12.5ul 2x TD buffer, 1.25ul transposase (Illumina, catalog nr 20034197), 8.25ul PBS, 0.25ul 1% Digitonin, 0.25ul 10% Tween-20, 2.5ul nuclease-free water (Invitrogen, catalog nr AM9937)) and incubated at 37ºC for 30min, 1000RPM. Transposed fragments were purified using a Zymo Clean and Concentrator5 kit (Zymo, catalog nr R1013), followed by amplification (Ultra II Q5, NEB, catalog nr M0544L) and double size selection of 100-1000bp fragments with AMPure XP beads (Beckman Coulter, catalog nr A63881). Library was sequenced using 2x50bp reads.