GSM7037200: sample.1.8gy_24hrs; Homo sapiens; RNA-Seq
Total RNA was isolated from 5 M Nalm6 cells by lysing with Trizol Reagent (Invitrogen Waltham, MA) followed by extraction with 24:1 chloroform:isoamyl alcohol (Sigma Aldrich St. Louis, MO). After separation by centrifugation, the aqueous phase was frozen in 100% isopropanol overnight, and then RNA was then precipitated and washed twice with 75% ethanol. RNA yield was quantified using an Epoch Microplate Spectrophotometer (Biotek Winooski, VT). From each isolate 10 µg of RNA was treated with 2 units of DNAse I for 10 minutes at 37 °C and purified on a Monarch RNA Cleanup Spin Column (New England Biolabs Ipswich, MA), all according to the manufacturer's instructions. Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 500ng to 1ug of total RNA. Ribosomal RNA was blocked using FastSelect reagents (Qiagen) during cDNA synthesis. RNA was fragmented in reverse transcriptase buffer with FastSelect reagent and heating to 94 degrees for 5 minutes, 75 degrees for 2 minutes, 70 degrees for 2 minutes, 65 degrees for 2 minutes, 60 degrees for 2 minutes, 55 degrees for 2 minutes, 37 degrees for 5 minutes, 25 degrees for 5 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.