GSM1881626: NAI (ESCs, Fibrillarin sh #1); Mus musculus; OTHER
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. Libraries for NAI-treated RNA were prepared with minor changes to the protocol used for CIRS-seq (Incarnato et al., 2014). Briefly, 2 μg of total RNA were denatured at 70°C for 5 minutes, then stored immediately on ice. RNA was treated with 100mM NAI for 20 minutes at 37°C with moderate shaking. Reaction was quenched by addition of 0.7M 2-Mercaptoethanol and purified on RNeasy Micro Spin columns (Qiagen). Reverse transcription was performed using 200U of SuperScript III Reverse Transcriptase, and random hexamers fused with the reverse complement of the Illumina TruSeq RNA 3’ Adapter (CCTTGGCACCCGAGAATTCCANNNNNN). The reverse transcription reaction was conducted in 50 minutes at 50°C, followed by 15 minutes at 75°C to inactivate the reverse transcriptase. 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 50-200nt 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. 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.
GEO Accession
GSM1881626