GSM7401846: THP1, CYR61 5, rep5; Homo sapiens; RNA-Seq
After incubation, we washed cells twice with PBS, then used TRIzol (ambion) and RNeasy Plus Mini Kit (Qiagen) to extract RNA following the manufacturer's recommended protocol. We determined total RNA integrity using an Agilent Bioanalyzer or 4200 Tapestation. Using 500 ng - 1 ug of starting total RNA, we removed ribosomal RNA using an RNase-H method using RiboErase kits (Kapa Biosystems). We fragmented mRNA in reverse transcriptase buffer and heated samples to 94 degrees for 8 minutes. We reverse transcribed mRNA to cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. We performed a second strand reaction to yield ds-cDNA. We blunt ended the cDNA, added an A base to the 3' ends, and then ligated Illumina sequencing adapters to the ends. We amplified the ligated fragments for 12-15 cycles using primers incorporating unique dual index tags. We sequenced fragments on an Illumina NovaSeq 6000 using paired end reads extending 150 bases.