title: GSM1881623: 2OMe-seq 1mM dNTPs (ESCs, Fibrillarin sh #2); Mus musculus; 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. For each experiment, 2 μg of total RNA were spiked-in with 100 ng of in vitro transcribed RNAs (where specified). 2OMe-seq libraries were prepared with minor changes to the protocol used for CIRS-seq (Incarnato et al., 2014). Briefly, reverse transcription was performed in a 20 μl reaction, using 1mM final dNTPs, 0.5U/μl AMV Reverse Transcriptase (NEB), and random hexamers fused with the reverse complement of the Illumina TruSeq RNA 3’ Adapter (CCTTGGCACCCGAGAATTCCANNNNNN). 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 recovery, and excess adapter removal was performed using Agencourt Ampure XP beads (Beckman Coulter). Following cleanup, cDNAs were separated on a 10% TBE-Urea PAGE gel, and a gel slice corresponding to 70-300nt fragments 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. This approach allowed us to keep the strand-specificity of the library, so that each read started 1 nt downstream of the RT stopping point. The adapter-ligated cDNAs were then subjected to 15 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: GSM1881623