GSM8192431: CAGEseq_H2228 shNIPBL_#2 5D rep1; Homo sapiens; OTHER
Cells were lyzed directly on culture dish with TRIzol RNA isolation reagent (Thermo fisher scientific). Total RNA was extracted by thorough mixing with chloroform and isopropanol precipitation of upper aqueous phase. 5 µg total RNA was reverse transcripted using a primer that carried a 15-nt random region and EcoP15I recognition sequence and SuperScriptIII RNase H- reverse transcriptase (Thermo fisher scientific) in the presence of trehalose/sorbitol to increase efficiency of reverse transcription. Diol groups on RNA/cDNA duplex were oxidated and labelled with biotin hydrazide (Vector laboratories). RNase I (Promega) treatment was performed to cleave single-stranded RNA regions that were not hybridized with first-strand cDNA. tRNA-coated streptavidin beads (Thermo fisher scientific) were used to capture biotinylated RNA/cDNA duplex. Sample was washed three times with wash buffer A [4.5 M NaCl, 50 mM EDTA (pH 8.0) and 0.1 % Tween20], once with wash buffer B [10 mM Tris–HCl (pH 8.5), 1 mM EDTA (pH 8.0), 0.5 M NaOAc (pH 6.1) and 0.1 % Tween20] and once with wash buffer C [0.3 M NaCl, 1 mM EDTA (pH 8.0) and 0.1 % Tween20]. Single-stranded cDNAs were released from streptavidin beads by heating at 95 °C for 5 min. Released cDNA was treated with RNase H (Thermo fisher scientific) and RNase I (Promega) at 37 °C for 45 min and proceed for library construction. Library was constructed with modified primers for compatible sequencing on Illumina NextSeq/NovaSeq sequencer. Ligation of bar-coded 5' linker was performed. Second-strand cDNA was synthesized using Deep Vent (exo-) DNA polymerase (NEB) and biotinylated second-strand primer. cDNA was treated with shrimp alkaline phosphatase (Thermo fisher scientific) to remove phosphate group from 5' linker. EcoP15I (NEB) was used to cleave 27 nt downstream of the recognition site at the 5' linker. 3' linker was ligated to the 27-nt-long tags, purified with tRNA-coated streptavidin beads and amplified with 2x Phusion high-fidelity PCR mix (NEB). CAGE libraries were treated with Exonuclease I (NEB) to degrade any single-stranded DNA before sequencing. Libraries were sequenced on NextSeq/NovaSeq using SR50 to obtain around 40 M reads for each library.