GSM8921774: AKTP MTO, vehicle control, replicate 1; Mus musculus; ATAC-seq
Dissociation was done in 1.5 ml 1:10 TryplE medium for 30 min at 37 ºC with vigorously pipetted every 10 min, quenched in Advanced DMEM/F12 (Gibco, 12634028), supplemented with 10mM HEPES (Gibco, 11560496), Glutamax (Gibco, 11574466), and quickly centrifuged for 5 min at 500 g at 4 ºC. The pellet was washed in cold PBS and cells were lysed in 300 μl cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2 in water) containing 0.1% IGEPAL CA-630 (Sigma, l8896) following centrifugation for 10 min at 500g at 4 ºC. 200 μl of supernatant was aspirated and centrifugation was repeated for 5 min. Counting was done in RSB with a Neubauer chamber and aliquots of 50.000 nuclei were prepared and resuspended in 50 μl transposition mix (25 μl 2x TD buffer (Illumina, 20034197), 2.5 μl transposase (Illumina, 20034197), 16.5 μl PBS, 0.5 μl 1% digitonin (Millipore, 300410), 0.5% 10% Tween-20 (Sigma-Aldrich, P9416) and 5 μl water) by pipetting up and down 10 times. Transposition reactions were incubated at 37 ºC for 30 min in a thermomixer and then cleaned up with Qiagen MinElute PCR Purification Kit (Qiagen, 28004). Libraries were prepared as described in Buenrostro et al., 2015. Library concentration and profile were assessed with Qubit HS dsDNA (Invitrogen, Q33321) and Tapestation (Agilent, 5067-5589, 5067-5590), respectively, and sequencing was done with Illumina NextSeq 2000 2x100 bp PE sequencing for at least 200 M reads.