Name: GSM8306486
Instrument: Illumina NovaSeq 6000
Strategy: ATAC-seq
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: At the time point after washout or mock washout, cells were rinsed in PBS, and harvested using 0.25 % trypsin with GSI and/or BRM014 according to the sample, and quenched with SC2 media containing GSI and/or BRM014. Cells were counted, and 100,000 cells were transferred to an Eppendorf tube, and combined with 10,000 fly spike-in cells. The spike-in S2 cells had been collected, resuspended in BAMBANKER cryopreservative media (VWR 101974-112), aliquoted, and stored at -80°C. All spike in aliquots for one experimental time course replicate were thawed and combined before beginning the time course. ATAC was performed according to the OmniATAC protocol. Briefly, cells were pelleted at 500 g for 5 min at 4°C, and supernatant was removed. The cell pellet was resuspended in 50 mL ATAC lysis buffer by pipetting up and down 3 times (Cold ATAC-Resuspension Buffer (10 mM Tris–HCl pH 7.5, 10 mM NaCl and 3 mM MgCl2) + 0.1 % NP40 (EMD Millipore 492018) + 0.1 % Tween-20 (EMD Millipore 655206) + 0.01 % digitonin (Promega G9441)). Up to 4 samples were processed at 1 time, and samples were staggered by 15 seconds. After exactly 3 min of lysis, 1 mL of ATAC wash buffer (Cold ATAC-Resuspension Buffer + 0.1 % Tween-20 (EMD Millipore655206)) was added to the tube. Nuclei were pelleted at 500 g for 10 min at 4°C, and supernatant was removed. Nuclei were resuspended in 50 mL of Transposition mix (1x TD buffer (Illumina 20334197), 3 mL Tn5 transposase (Illumina 20334197), 0.33x PBS, 0.01 % digitonin, 0.1 % Tween-20), and incubated at 37°C in a thermomixer at 1000 rpm for 30 minutes. The reactions were quenched with 250 mL of binding buffer from the MinElute PCR purification kit (Qiagen 28004) and purified using this kit. The eluate was then used as the input for barcoding PCR, to add sequencing adapters and sequencing barcodes, as described in Grandi et al.. Both the qPCR and Qubit methods were used to quantify the libraries to determine the total number of PCR cycles, as described in the OmniATAC protocol. After the final PCR cycles, libraries were cleaned up with the MinElute PCR purification kit, quantified using the NEBNext Library Quant kit (New England Biolabs E7630), and pooled for sequencing. Libraries were sequenced paired end on a NovaSeq with an S1 single lane 100 cycle kit.