title: GSM7913282: k562, WTHYPB, rep10; Homo sapiens; OTHER
library_construction_protocol: Total RNA was isolated from every three replicates using the RNEasy Plus Mini Kit following manufacturer's instructions. RNA was eluted in 40ul RNase-free water and quantified using the Qubit RNA HS Assay Kit. We performed first strand synthesis on each replicate with Maxima H Minus Reverse Transcriptase. We mixed 2ug of total RNA with 1ul of 10mM dNTPs and 1ul of 50uM SMART_dT18VN primer, brought the total volume up to 14ul, and incubated it at 65C for 5 minutes. After a 1 minute incubation on ice, we added 4ul RT Buffer, 1ul RNaseOUT, and 1ul of Maxima H Minus Reverse Transcriptase. The solution was mixed by pipetting and incubated at 50C for 1 hour followed by heat inactivation at 85C for 10 minutes. First strand cDNA synthesis was performed by reverse transcribing polyA mRNAs using an oligo(dT) primer that also adds on a common primer sequence. Self-reporting transcripts (SRTs) were amplified by PCR. PCR products were tagmented and indexed using the Illumina Nextera XT library preparation kit. For detailed protocol, see bioRvix (https://www.biorxiv.org/content/10.1101/2023.06.07.544098v1)