title: GSM8483679: Mouse_FNRMS_ATACseq_vehicle_2; Mus musculus; ATAC-seq
library_construction_protocol: Cells were collected and washed with ice-cold PBS, 50.000 cells were pelleted in a pre-cooled 1.5 ml tube by centrifugation for 5 minutes at 600 rcf at 4 ºC in a microcentrifuge. The supernatant was removed and 50 µl of ATAC-lysis buffer was added (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40) and the nuclei were pelleted for 10 minutes at 600 rcf at 4 ºC in a microcentrifuge. Subsequently, the supernatant was removed and nuclei were resuspended in 50 µl transposition reaction buffer (27.5 µl H2O, , 25 µl TD buffer, 2.5 µl Tagment DNA Enzyme 1) (Tagment DNA Enzyme and Buffer Small Kit, Illumina, 20034197) and incubated for 30 minutes at 37 ºC at 1000 rpm in a thermomixer. The transposed DNA was cleaned up immediately following transposition using the ZymoDNAClean & Concentrator-5 kit (Zymo, WA, USA, D4014) and amplified in one pre-amplification step (5 cycles) and a final amplification (5 to 7 cycles) in the same 50 µl reaction using 1x NEBNext High-fidelity PCR master mix (New England Biolabs, MA, USA, M0541S). Following amplification, the libraries were purified using the ZymoDNAClean & Concentrator-5 kit (Zymo, D4014) followed by 0.55x-1.75x dual size selection using AMPure XP beads (Beckman Coulter, CA, USA). The quality of the libraries was assessed using an Agilent Bioanalyzer 2100 with the High Sensitivity DNA analysis kit (Agilent Technologies, 5067-4626) and the concentration was determined using Qubit™ dsDNA HS Assay kit (Invitrogen, Q32854). The libraries were diluted to 4 nM and pooled together for SE50 sequencing on an Illumina HiSeq4000, aiming for minimally 25 million mapped and unique reads.