GSM1881617: Alkaline hydrolysis (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 were resuspended in 50mM Na2CO3 buffer, and heated 6 minutes at 95°C. Fragmented RNA was purified on RNA Clean & Concentrator™-5 columns (Zymo Research). The eluted RNA was dephosphorylated by treating with 5U Antarctic Phosphatase (NEB) at 37°C for 30 minutes, followed by 10 minutes at 65°C to inactivate the enzyme. Dephosphorilated RNA fragments were 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, RNA fragments were purified on RNeasy Micro Spin columns (Qiagen) to remove the excess adapter, eluted, and then ligated to the Illumina TruSeq RNA 5’ Adapter (GUUCAGAGUUCUACAGUCCGACGAUC) using 20U of T4 RNA Ligase 1 (NEB), by incubating at 25°C for 2 hours. The adapter-ligated RNA was purified again on RNeasy Micro Spin columns (Qiagen) to get rid of the excess adapter, and reverse transcription was performed in a 20 μl reaction, using 200U SuperScript III Reverse Transcriptase (Invitrogen), and an oligonucleotide complementary to the 3’ adapter (AGACGTGTGCTCTTCCGATCT). 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. cDNA fragments 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 cDNAs were 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
GSM1881617