GSM4977360: CTL-isoalloLCA rep4; Mus musculus; ATAC-seq
20,000 cells were sorted in 1.5ml DNA lo-bind tubes, wash with PBS, and then lysed as well as tagging by Tn5 transposase mixture: 1x Tagment DNA Buffer, 0.5uL Tagment DNA Enzyme (Nextera DNA Library Preparation Kit, Illumina) and 0.2mg/ml digitonin (#G9441, Promega) on ice. Cells were incubated at 37°C for 30 minutes with agitation followed by DNA purification using the MinElute Reaction Cleanup Kit (QIAGEN, Hilden, Germany). ATAC-seq libraries were constructed through first-round PCR using the NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs, MA, USA) with primers (0.5uM each) from (Buenrostro et al., 2015) with the following thermal cycles: 5 minutes at 72°C, 30 s at 98°C, followed by 7 cycles [98°C for 10 s, 63°C for 30 s and 72°C for 60 s] and a final extension at 72°C for 5 minutes. PCR products went through 2 rounds of size selection by Agencourt AMPure XP beads (Beckman Coulter) (0.65x and 1.8x volume to remove long and short fragments respectively) and eluted in 18uL of EB (QIAGEN). Second-round PCR was performed using the NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs, MA, USA) with primers (1.25uM each) with the following thermal cycles: 30 s hot-start at 98°C, followed by 7∼13 cycles [98°C for 10 s, 63°C for 30 s and 72°C for 60 s] and a final extension at 72°C for 5 minutes. The final PCR products were purified by Agencourt AMPure XP beads (x1.8 vol.), quantified by tapestation and qPCR, pooled and sequenced as paired-end (38+37bp) on an Illumina NextSeq 500 instrument in high-output mode.