GSM8048709: RNA_NTC_2, Mono; Homo sapiens; RNA-Seq
Cells were washed in 50 µl cold PBS and centrifuged at 1200 rpm for 4 min. The cell pellet was resuspened in 50 µl of cold lysis buffer (48.5 µl of resuspension buffer (10 mM Tris-HCl pH 7.5 (15567-027, Thermo Fisher Scientific), 10 mM NaCl, 3 mM MgCl2), 0.5 µl 10% NP-40, 0.5 µl 10% Tween-20 (P1379, Sigma-Aldrich), and 0.25 µl 2% Digitonin (G9441, Promega)) and incubated for 3 min on ice. Wash buffer (990 µl of resuspension buffer, 10 µl 10% Tween-20) was added to the cell lysate and centrifuged at 1000 xg at 4°C for 5 min. The cell pellet was resuspened in transposition reaction mix (25 µl TD Buffer, 16.5 µl PBS, 0.5 µl 10% Tween-20, 0.25 µl 2% Digtonin, 2.5 µl Tn5, 5 µl H2O) and incubated at 1000 rpm at 37°C for 30 min. DNA was purified by using MinElute PCR Purification Kit (Qiagen), eluated in 20 µl elution buffer, and stored at -20 °C until sequencing. RNA and library preparation integrity were verified with LabChip Gx Touch 24 (Perkin Elmer). 0.3-1µg of total RNA was used as input for SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian (Takara Bio). Sequencing was performed on the NextSeq2000 instrument (Illumina) using with 1x72bp single end setup.