GSM6327086: Treg, Il10 negative, rep2 [ATAC_G2_neg]; Mus musculus; ATAC-seq
Combined protocol for extraction and library preparation, see below After sorting, cells were pelleted in a fixed rotor benchtop centrifuge at 500g for 5 minutes at 4°C. Cells were then washed in 1ml cold 1x PBS and pelleted again. Supernatant was aspirated and cells were resuspended in 50µl ice-cold cell lysis buffer (10mM Tris-Cl pH 7.4; 10mM NaCl; 3mM MgCl2; 0.1% NP-40) to disrupt plasma membranes. Nuclei were pelleted at 1000g for 10 minutes. Supernatant was aspirated and nuclei were resuspended in 50µl transposition reaction mixture (25µl TD buffer; 2.5µl TDE1; 22.5µl ddH2O; llumina, 20034197). Samples were incubated in a ThermoMixer at 1100rpm for 45 minutes at 42°C. DNA was then purified using a MinElute PCR Purification Kit (QIAGEN, 28004), per manufacturer instructions. DNA was eluted in 10µl buffer EB. Libraries were then barcoded and amplified with NEBNext High-Fidelity Master Mix and primers from (Buenrostro et al., 2013) (50µl reaction with 10µl DNA and 2.5µl of 25µM primers – 1 cycle of 5min at 72°C, 30sec at 98°C; 5 cycles of 10sec at 98°C, 20sec at 63°C, 1min at 72°C). A qPCR on the product determined that an additional 7 cycles (10sec at 98°C, 20sec at 63°C, 1min at 72°C) were required. Library was purified and size selected with AMPure beads (Beckman Coulter, A63881): 45µl of PCR product was incubated with 18µl beads and supernatant was collected (beads bound larger >2000bp fragments). Supernatant (63µl) was then incubated with an additional 63µl of beads for 5 minutes, supernatant was removed, beads were washed 2x with 75% ethanol, and DNA was eluted into 50µl H2O by incubating for 2 minutes. After quantification and quality control by Agilent BioAnalyzer, pooled libraries were run on a HiSeq 4000 in a PE50 run, using the HiSeq 3000/4000 SBS Kit (Illumina). The loading concentration was 3nM and a 5% spike-in of PhiX was added to the run to increase diversity and for quality control purposes. The run yielded average 36M reads per sample.