GSM7988660: AG25367_g2_CTL_d4; Homo sapiens; RNA-Seq
Total RNA was extracted with TRI reagent (Millipore Sigma) and treated with DNase I (Qiagen) using a RNeasy MinElute Cleanup Kit (Qiagen). RNA integrity was determined using Agilent 4200 TapeStation. Library preparation was performed with 1 µg of total RNA. Ribosomal RNA was removed by an RNase-H method using the RiboErase kit (Kapa Biosystems). mRNA was then fragmented in reverse transcriptase buffer and heating to 94°C for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III reverse transcriptase enzyme and random hexamers (Life Technologies) per manufacturer's instructions. A second strand reaction was performed to yield double stranded 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 targeting 30M clusters.