GSM5799838: AsPC-1_shARG2 #2_RNA_rep2; Homo sapiens; RNA-Seq
RNA-seq: Total RNA was isolated and purified using miRNeasy Mini Kit (Qiagen, 217004). ATAC-seq: Cells were resuspended in 50 μl ice-cold ATAC lysis buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% (v/v) Igepal CA-630) and centrifuged at 500 g at 4°C to isolate nuclear pellets that were then treated in 50 μl reactions with Nextera Tn5 Transposase (Illumina, FC-121-1030) for 30 min at 37°C. RNA-seq: mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. ATAC-seq: Transposed DNA was purified using a PCR purification kit (Zymo research, D4034) and then amplified immediately in 50 μl reactions with high-fidelity 2X PCR Master Mix (New England Biolabs) using a common forward primer and different reverse primers with unique barcodes for each sample. From the reaction mix, 45 μl was kept on ice after 5 cycles of PCR, while 5 μl was amplified by qPCR for 20 additional cycles; the remaining 45 μl was then amplified for the 5-7 cycles required to achieve 1/3 of the maximum qPCR fluorescence intensity. Amplified DNA was purified over columns and primer dimers (<100 bp) were removed using AMPure beads (Beckman Coulter).