GSM9139528: Si-1; Homo sapiens; RNA-Seq
HCT116 cells in the logarithmic growth phase were treated with either negative control siRNA (NC) or siCPT2 for 48 hours. The cells were collected, washed three times with PBS, and lysed with 1 mL of TRIzol for 5 minutes on ice. (1) mRNA was isolated by Oligo(dT)-attached magnetic beads. (2) mRNA was then randomly fragmented in fragmentation buffer. (3) First-strand cDNA was synthesized with fragmented mRNA as template and random hexamers as primers, followed by second-strand synthesis with addition of PCR buffer, dNTPs, RNase H and DNA polymerase I. Purification of cDNA was processed with AMPure XP beads. (4) Double-strand cDNA was subjected to end repair. Adenosine was added to the end and ligated to adapters. AMPure XP beads were applied here to select fragments within size range of 300-400 bp. (5) cDNA library was obtained by certain rounds of PCR on cDNA fragments generated from step 4.