GSM6217121: NGT CTL 36h i01; Homo sapiens; RNA-Seq
Cells were lysed and RNA extracted using Trizol Reagent (Life Technologies; Cat #15596-018) and miRNAeasy kit (Qiagen; Cat #217004). RNA was checked for quality using the Agilent RNA 600 nano kit and Bioanalyser instrument (Agilent Technologies, Santa Clara, CA). Aliquots of RNA (1 µg) were analyzed using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold protocol (Illumina). Ribosomal RNA was removed from the sample using 35 µl rRNA removal beads (Illumina) on a magnetic plate followed by clean-up of the ribosomal-depleted RNA with 193 µl Agencourt RNAClean XP beads (Beckman Coulter), 70% ethanol wash and elution into 10 µl Elution buffer (Illumina). The RNA sample was fragmented for 4 min at 94°C in Elute, Prime, Fragment High Mix (Illumina) and then subjected to first strand cDNA synthesis with 1 µl Superscript III reverse transcriptase (Life Technologies) per sample using a thermocycler programmed to 25°C for 10 min, 50°C for 15 min and 70°C for 15 min. Second strand cDNA was synthesized by addition of Second Strand Marking Master Mix and samples were incubated at 16°C for 60 min. Samples were subject to another bead clean up prior to A-tailing and ligation of adapters as per kit instructions (Illumina). Following a third bead clean-up, samples were enriched for DNA fragments by amplification using the Illumina PCR Primer Cocktail and PCR Master Mix, subjected to 98°C for 30 min, followed by a pre-defined cycle (98°C for 10 s, 60°C for 30 s and 72°C for 30 s) that was repeated 3-15 times, based on each individual sample, and finally incubated for 5 min at 72°C. Samples were cleaned, and validated for DNA concentration using the Qubit dsDNA HS assay kit (Invitrogen) and for base pair size and purity using the Agilent High Sensitivity DNA chip and Bioanalyser instrument. Libraries were subjected to 100-bp paired-end sequencing on the X Ten platform (Illumina) at the Beijing Genomics Institute (BGI), Hong Kong, China. Approximately 18 million reads/sample were assigned to genes with 16,229 genes surviving the expression threshold.