title: GSM1881615: 2OMe-seq 1mM dNTPs (HeLa, Method #2); Homo sapiens; OTHER
library_construction_protocol: Cells were washed twice in 1X PBS, and lysed by addition of 1ml ice-cold TRIzol (Invitrogen). RNA integrity measurements were performed using Fragment Analyzer™ (Advanced Analytical). All samples had RNA Quality Number (RQN) greater than 9.8. Briefly, 2 μg of total RNA were fragmented by addition of 5X First Strand Buffer (Invitrogen), and incubation at 95°C for 5 minutes. Fragmented RNA was purified on RNA Clean & Concentrator™-5 columns (Zymo Research). The eluted RNA was end repaired by adding 20U of T4 PNK (NEB), and incubating at 37°C for 1h, and then purified again on RNA Clean & Concentrator™-5 columns. A pre-adenylated (rApp) adapter (AGATCGGAAGAGCACACGTCT) was ligated to the end-repaired RNA fragments using 400U of T4 RNA Ligase 2, Deletion Mutant (Epicentre) in the presence of 20% PEG-8000, by incubating at 16°C for 18 hours. The day after, reactions were purified on RNeasy Micro Spin columns (Qiagen) to get rid of the excess adapter. Following cleanup, adapter-ligated RNA fragments were separated on a 10% TBE-Urea PAGE gel, and a gel slice corresponding to 200-250nt fragments was cut. RNA was recovered by passive diffusion in RNA Diffusion Buffer [450mM Ammonium Acetate, 0.05% SDS] for 16 hours at 4°C with end-to-end rotation. Following isopropanol precipitation, reverse transcription was performed in a 20 μl reaction, using 1mM final dNTPs (High dNTP sample), 0.5U/μl AMV Reverse Transcriptase (NEB), and an oligonucleotide complementary to the 3’ adapter (AGACGTGTGCTCTTCCGATCT). The reverse transcription reaction was conducted in 30 minutes at 42°C, followed by 10 minutes at 95°C to inactivate the AMV enzyme. Template RNA was degraded by addition of 1 μl of Ribonuclease H (Ambion), and 1 μl of RNase Cocktail Enzyme Mix (Ambion), followed by 30 minutes incubation at 37°C. cDNAs were purified on RNA Clean & Concentrator™-5 columns (Zymo Research), separated on a 10% TBE-Urea PAGE gel, and a gel slice corresponding to 50-150nt fragments (corresponding to truncated reverse transcription products) was cut. DNA was recovered by passive diffusion in TE Buffer [10mM Tris-HCl, 1mM EDTA] for 16 hours at 37°C with moderate shaking. Following ethanol precipitation, an adapter corresponding to the reverse complement of the standard Illumina TruSeq Small RNA 5’ Adapter (GATCGTCGGACTGTAGAACTCTGAAC), modified with a 5’-P group and a 3’-C3 spacer, was ligated to cDNAs 3’-OH termini using 200 U of CircLigase II for 6 hours at 65°C. The adapter-ligated cDNAs were then subjected to 18 cycles of PCR using standard Illumina TruSeq primers, and excess primers were removed using Agencourt Ampure XP beads. Libraries were pooled in equimolar amounts, and subjected to sequencing on the Illumina™ NextSeq 500 Sequencer.
attributes.tag: GEO Accession
attributes.value: GSM1881615