GSM1881618: RNase H (HeLa); Homo sapiens; 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. Briefly, 2 μg of total RNA and 1 μl of a 10μM random pentadecamers (NNNNNNNNNNNNNNN) mixture were heated 1 minute at 95°C, and annealed by slowly decreasing the temperature to 25°C. Reverse transcription was performed using 200U of SuperScript III Reverse Transcriptase (Invitrogen) by incubating at 50°C for 50 minutes, followed by 15 minutes at 75°C to inactivate the enzyme. Reaction volume (20 μl) was then brought to 100 μl with RNase H Digestion Buffer [20mM HEPES pH 8.0, 50mM KCl, 4mM MgCl2, 1mM DTT, 50 μg/ml BSA], supplemented with 20U Ribonuclease H (Ambion), and incubated for 1 hour at 37°C. DNA was degraded by adding 50 U of DNase I (Thermo Scientific) for 15 minutes at 37°C, and RNA was recovered by TRIzol (Invitrogen) extraction. 5’-P RNA fragments derived from RNase H digestion were ligated to the Illumina TruSeq RNA 5’ Adapter (GUUCAGAGUUCUACAGUCCGACGAUC) using 20U of T4 RNA Ligase 1 (NEB) in the presence of 20% PEG-8000, by incubating at 16°C for 18 hours. The adapter-ligated RNA was recovered by ethanol precipitation, and subjected to reverse transcription 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 80-200nt 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, the adapter-ligated DNA was 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
GSM1881618