title: GSM9073004: shTRUB2_1 HCT116 _BACS; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated using TRIzol (Invitrogen) and Direct-zol RNA Miniprep Plus (Zymo Research) according to the manufacturer's protocol. Ribo– RNA was isolated using RiboMinus Eukaryote System v2 (Invitrogen) according to the manufacturer's protocol. PolyA+ RNA was isolated by two rounds of polyA-tailed selection using Dynabeads mRNA DIRECT Purification Kit (Invitrogen) according to the manufacturer's protocol. To remove genomic DNA contamination, RNA was then treated with Turbo DNase and purified by Zymo-IC Column with RNA binding buffer. 50–100 ng ribo– or polyA+ RNA was fragmented by NEBNext Magnesium RNA Fragmentation Module at 94 °C for 4 min according to the manufacturer's protocol and purified by Zymo-IC Column with RNA binding buffer. The fragmented RNA was mixed with 5 μl 10× T4 PNK reaction buffer (NEB), 5 μl T4 PNK (NEB), and 2.5 μl SUPERase•In RNase Inhibitor (Invitrogen) in a 50 µL final solution and incubated at 37 °C for 1 h. The 3'-repaired RNA was purified by Zymo-IC Column with RNA binding buffer and eluted with 10 μl nuclease-free H2O. The eluted RNA was then mixed with 1 μl synthetic 30mer spike-ins (2%) and 1 μl of 20 μM RNA adapter (5'-/5rApp/AGATCGGAAGAGCGTCGTG/3SpC3/-3'), incubated at 70 °C for 2 min and immediately placed on ice. Next, 2.5 μl 10× T4 RNA Ligase reaction buffer (NEB), 1 μl SUPERase•In RNase Inhibitor, 7.5 μl 50% PEG 8000 (NEB), and 2 μl T4 RNA Ligase 2, truncated KQ (NEB) were added to the mixture and the reaction was incubated at 25 °C for 2 h followed by 16 °C for 14 h. To digest excess adapters, the solution was further diluted to 47 μl with nuclease-free H2O and treated with 2 μl 5'-Deadenylase (NEB) at 30 °C for 1 h followed by adding 1 μl RecJf (NEB) and incubating at 37 °C for 1 h. The 3'-ligated RNA was purified by Zymo-IC Column with RNA binding buffer and eluted with 10 μl nuclease-free H2O. A 7 μl aliquot was subjected to BACS library construction, while the rest 3 μl was saved as control sample and diluted to 12.5 μl with nuclease-free H2O. For BACS, 1 M 2-bromoacrylamide (Enamine) was prepared by dissolving the solid in DMSO. 7 μl 3'-ligated RNA was added into a 20 μl solution containing 250 mM 2-bromoacrylamide and 625 mM phosphate buffer (pH 8.5) and incubated at 85 °C for 30 min. The treated RNA was double purified by Micro Bio-Spin P-6 Tris Column (Bio-Rad) and Zymo-IC Column with RNA binding buffer and finally eluted with 12.5 μl nuclease-free H2O. Both treated and control RNA samples were mixed with 1 μl of 2 μM RT primer (5'-ACACGACGCTCTTCCGATCT-3') and 1 μl of 10 mM dNTP mix (NEB), incubated at 70 °C for 2 min and immediately placed on ice. Next, 4 μl 5× Maxima H– RT buffer (Thermo), 0.5 μl RiboLock RNase Inhibitor (Thermo), and 1 μl Maxima H– Reverse Transcriptase (Thermo) were added to the mixture and the reaction was incubated at 50 °C for 1 h. To digest excess RT primers, the solution was treated with 1 μl Exo I (NEB) and incubated at 37 °C for 30 min followed by adding 1 μl of 0.5 M EDTA (Sigma) to quench the reaction. To hydrolyze the RNA, 2.5 µl of 1 M NaOH (Sigma) was added and the solution was then incubated at 70 °C for 12 min followed by adding 2.5 µl of 1 M HCl (Sigma) to neutralize NaOH. The cDNA was finally purified with Dynabeads MyOne Silane (Invitrogen) and eluted with 13 µl nuclease-free H2O. The eluted cDNA was then mixed with 2 µl of 25 μM cDNA adapter (5'-/5Phos/NNNNNNAGATCGGAAGAGCACACGTCTG/3SpC3/-3'), incubated at 70 °C for 2 min and immediately placed on ice. Next, 5 μl 10× T4 RNA Ligase reaction buffer, 25 μl 50% PEG 8000, 0.5 μl of 100 mM ATP (NEB), 3.5 μl DMSO (Thermo), and 1 μl T4 RNA Ligase 1, high concentration (NEB) were added to the mixture and the reaction was incubated at 25 °C for 16 h. The ligated cDNA was purified with Dynabeads MyOne Silane and eluted with 15 µl nuclease-free H2O. The eluted DNA was amplified with NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) and NEBNext Ultra II Q5 Master Mix for 10 cycles according to the manufacturer's protocol. The PCR products were purified with 0.8× AMPure XP beads and quantified with Qubit dsDNA HS Assay Kit (Thermo) according to the manufacturer's protocol. BACS and control libraries were sequenced on NextSeq 2000 (60 bp paired end) with no PhiX added.