title: GSM8000249: RNA seq library of K562 cells expressing control shRNA Batch2 Rep1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNAs were purified from cells using TRIzol LS Reagent (Invitrogen) following manufacturer's instructions, treated with DNase (QIAGEN or Invitrogen) and cleaned up using RNeasy Mini kit (QIAGEN). For ATAC-seq, 1 x 105 cells were collected, washed with PBS, and pelleted by centrifugation at 500 xg, 4 oC for 5 min. Cells were then lysed with 50 uL lysis buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin) on ice for 3 min and neutralize with 250 uL RSB buffer with Tween-20 (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20). For RNA-seq, 1 ug total RNA (RNA integrity >= 9.9) were used for stranded mRNA library preparation using the Illumina TruSeq® Stranded mRNA Library Prep kit. The RNA libraries were indexed using the Illumina TruSeq RNA UD Indexes. For ATAC-seq, 1 x 105 cells were collected, washed with PBS, and pelleted by centrifugation at 500 xg, 4 oC for 5 min. Cells were then lysed with 50 uL lysis buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin) on ice for 3 min and neutralize with 250 uL RSB buffer with Tween-20 (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20). Nuclei were counted on a hemocytometer and 5 x 104 nuclei underwent tagmentation. For tagmentation, per 50 uL contained 25 uL 2x TD Buffer, 8.25 uL PBS, 0.5 uL 1% Digitonin, 0.5 uL 10% Tween-20, 2.5 uL Tn5 enzyme (Illumina, 2.5 uM) and 13.25 uL water. The reaction was incubated at 37oC for 30 min. DNA was purified using the Clean and Concentrate kit (Zymo) and eluted in 10 uL EB Buffer (QIAGEN). All or half of the eluted DNA was used for qPCR. In each reaction, 10 uL tagmented DNA was mixed with 25 uL 2x NEBNext High-Fidelity 2X PCR Master Mix (NEB), 2.5 uL i5 primer, 2.5 uL i7 primer, 0.25 uL 100X SYBR Green (Invitrogen) and 9.75 uL water. The qPCR reaction was performed as follows: 72oC 5 min, 98 oC 30 s; 98 oC 10 s, 63 oC 30 s, 72 oC 1 min. The reaction was stopped after 7 cycles. Libraries were pooled, purified with AMPure XP beads, and sequenced on an Illumina NextSeq 2000 sequencer.