GSM3144743: OmniATAC__MM057_Sox10_KD_72h; Homo sapiens; OTHER
OmniATAC-seq was performed as described previously [Corces et al., 2017]. Cells were washed, trypsinised, spun down at 1000 RPM for 5 min to remove the medium and resuspended in 1 mL. Cells were counted and experiments were only continued when a viability of above 90% was observed. 50,000 cells were pelleted at 500 RCF at 4°C for 5 min, medium was carefully aspirated and the cells were washed and lysed using 50 uL of cold ATAC-Resupension Buffer (RSB) (see Corces et al., 2017 for composition) containing 0.1% NP40, 0.1% Tween-20 and 0.01% digitonin by pipetting up and down three times and incubating the cells for 3 min on ice. The lysis was washed out by adding 1 mL of cold ATAC-RSB containing 0.1% Tween-20 and inverting the tube three times. Nuclei were pelleted at 500 RCF for 10 min at 4°C, the supernatant was carrefully removed and nuclei were resuspended in 50 uL of transposition mixture (25 uL 2x TD buffer (see Corces et al., 2017 for composittion), 2.5 uL transposase (100 nM), 16.5 uL DPBS, 0.5 uL 1% digitonin, 0.5 uL 10% Tween-20, 5 uL H2O) by pipetting six times up and down, followed by 30 minutes incubation at 37°C at 1000 RPM mixing rate. Samples were cleaned-up by MinElute. After MinElute clean-up and elution in 21 uL elution buffer, the transposed fragments were pre-amplified with Nextera primers by mixing 20 uL of transposed sample, 2.5 uL of both forward and reverse primers (25 uM) and 25 uL of 2x NEBNext Master Mix (program: 72°C for 5 min, 98°C for 30 sec and 5 cycles of [98°C for 10 sec, 63 °C for 30 sec, 72°C for 1 min] and hold at 4°C). To determine the required number of additional PCR cycles, a qPCR was performed [see Bruenrostro et al., 2015 for the determination of the number of cycles to be added]. The final amplification was done with the additional number of cycles, samples were cleaned-up by MinElute and libraries were further prepped using the KAPA Library Qunatificaton Kit as previously described [Corces et al., 2017].
GEO Accession
GSM3144743