title: GSM5662303: sh15.5K-1+Dox rep1; Homo sapiens; ncRNA-Seq
library_construction_protocol: Total RNA was isolated from cells using RNAzol. RNA was treated with RQ1 DNase (Promega) and purified by RNA Clean & Concentrator-5 (Zymo Research). The samples were then ligated to 3' randomized RNA adaptor (/rApp/NNNNTGGAATTCTCGGGTGCCAAGG/3SpC3/) with T4 RNA ligase 2 truncated KQ (NEB) in 1× T4 RNA ligase reaction buffer supplemented with 12.5% PEG 8000 at 16 °C for 18 h. The excessive adaptors were digested with 100 U 5' Deadenylase (NEB) at 30 °C for 1 h, incubated with 2 μg E. coli Single-strand DNA-binding protein (Promega) on ice for 30 min and followed by adding 60 U RecJf (NEB) for ssDNA digestion at 37 °C for another hour. The ligated RNA was ligated to 5' randomized RNA adaptor (GUUCAGAGUUCUACAGUCCGACGAUCNNNN) using T4 RNA Ligase 1 (NEB) in 1× T4 RNA ligase reaction buffer supplemented with 1 mM ATP at 16 °C for 18 h. The ligated RNA was column-purified by RNA Clean & Concentrator-5 (Zymo Research) and subjected to RNase H-based ribosomal RNA depletion according to the standard protocol with some modification5. For rRNA probe digestion, DNase I was replaced by RecJf exonuclease to prevent the digestion of 3' randomized RNA adaptor. After column-based purification, the rRNA-depleted RNA was reverse-transcribed using SuperScript IV reverse transcriptase (Invitrogen) in 1× RT buffer with truncated RT primer (GCCTTGGCACCCGAGAAT) to eliminate mispriming artifacts. The cDNA was amplified using Phusion High-Fidelity DNA Polymerase (Thermo Scientific) in 1× Phusion HF buffer (Thermo Scientific) with primers (RP1 forward primer and RPI-X reverse primer). The PCR-amplified cDNA was gel-purifed using a 4% NuSieve 3-1 Agarose gel (Lonza) to remove adapter dimers.