title: GSM5538167: HCT116_Cyto_rep4; Homo sapiens; ncRNA-Seq
library_construction_protocol: For subcellular RNA isolation, cells were trypsinized, centrifuged at 500 g for 3 min at 4 °C, washed with ice-cold PBS and recentrifuged. The cell pellet was resuspended in Hypotonic lysis buffer (10 mM Tris, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.3% NP-40 and 10% Glycerol) supplemented with 1 mM DTT, 100 U/mL RNase inhibitor and 400 μM RVC (NEB) and incubated on ice for 30 min. The resuspension was centrifuged at 1,000 g for 3 min at 4 °C. Approximately 70% volume of the supernatant was carefully removed without disturbing the nuclear pellet and precipitated by RNA precipitation solution (5% 3M sodium acetate, pH 5.5 and 95% ethanol), followed by RNA isolation using RNAzol; this was the cytoplasmic fraction. The remaining nuclear pellet was washed with Hypotonic lysis buffer three times. After centrifugation at 200 g for 2 min at 4 °C, the pellet was resuspended in Modified Wuarin-Schibler buffer (10 mM Tris, pH 7.0, 4 mM EDTA, 0.3 M NaCl, 1 M Urea and 1% NP-40) supplemented with 1 mM DTT, 100 U/mL RNase inhibitor and 400 μM RVC (NEB) and incubated on ice for 5 min. The sample was then centrifuged at 1,000g for 3 min at 4 °C and the supernatant was taken and precipitated by RNA precipitation solution, followed by RNA isolation using RNAzol; this was the nucleoplasmic fraction. For nucleoli isolation, the cell pellet volume was estimated for the next steps. The nuclear pellet was isolated and washed as described above. The pellet was resuspended in ten cell packed pellet volumes of ice-cold S1 buffer (0.25 M Sucrose, 10 mM MgCl2) supplemented with 1 mM DTT, 100 U/mL RNase inhibitor and 400 μM RVC (NEB) and then layered on ten cell packed pellet volumes of ice-cold S3 buffer (0.88 M Sucrose, 5 mM MgCl2) supplemented with 1 mM DTT, 100 U/mL RNase inhibitor and 400 μM RVC (NEB) and pelleted at 1,200g for 5 min at 4 °C. The pellet was then resuspended in ten cell packed pellet volumes of ice-cold S2 buffer (0.34 M Sucrose, 5 mM MgCl2) supplemented with 1 mM DTT, 100 U/mL RNase inhibitor and 400 μM RVC (NEB) and sonicated by using VibraCell Sonicator VCX130PB (Sonics & Materials, Inc.). The suspension was layered on ten cell packed pellet volumes of ice-cold S3 buffer (0.88 M Sucrose, 5 mM MgCl2) supplemented with 1 mM DTT, 100 U/mL RNase inhibitor and 400 μM RVC (NEB) and pelleted at 2,000g for 20 min at 4 °C. The supernatant was discarded, and the pellet was washed with S2 buffer and then centrifuged at 2,000g for 5 min at 4 °C. The pellet constituted the nucleolar fraction. 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.