Instrument: NextSeq 500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Cells were trypsinized, washed with cold PBS and resuspended in cold lysis buffer (10 mM Tris pH 7.4; 150 mM NaCl; 0.15% NP-40; RNase inhibitor). After 5 min incubation on ice, the suspension was layered over 1 ml sucrose buffer (10 mM Tris pH 7.4; 150 mM NaCl; 24% sucrose) and centrifuged (5 min, 3500 g). After removal of supernatant, nuclei pellet was briefly rinsed with cold PBS, resuspended in 250 μl Glycerol buffer (20 mM Tris pH 7.4; 75 mM NaCl; 0.5 mM EDTA; 50% Glycerol) and mixed with 250 μl nuclear lysis buffer (10 mM Tris pH 7.4, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M Urea, 1% NP-40) by vortexing for 4 sec. After 2 min incubation on ice, samples were centrifuged for 2 min at full speed. The remaining chromatin pellet was rinsed with cold PBS and resuspended in QIAzol lysis agent. RNA was extracted using QIAshredder and miRNeasy Mini Kit (Qiagen). After removal of rRNA by Ribo-Zero rRNA removal kit (Illumina), DNA libraries were generated using TruSeq Stranded Total RNA LT Sample Prep Kit and analyzed by NextSeq.