title: GSM8404071: K562_WholeCell_RNAseq_n1_(231128,240318_NK_hs_2100); Homo sapiens; RNA-Seq
library_name: GSM8404071
library_construction_protocol: RNA was extracted from whole cell K562 cells using Qiagen RNeasy Plus Mini Kit (Cat#74134) as per the manufacturer's instructions. First-strand synthesis was performed using Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) with the oligo(dT)-ME-B fusion oligonucleotide as the primer. Tagmentation was carried out using 100 ng of RNA-cDNA hybrids, ME-A loaded pAG-Tn5, and tagmentation buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2) for 1 hour at 37°C. The tagmented RNA-cDNA hybrids were purified using the HighPrep PCR Cleanup System (MagBio) at a 1x ratio following the manufacturer's instructions. Sequencing libraries were amplified using the NEBNext Master Mix (NEB) under the following PCR conditions: 58°C for 5 minutes, 72°C for 5 minutes, 98°C for 30 seconds, followed by 12 cycles of 98°C for 10 seconds and 60°C for 15 seconds, then 72°C for 2 minutes, and held at 4°C. Libraries were further purified using the HighPrep PCR Cleanup System (MagBio) at a 0.8x ratio according to the manufacturer's instructions. Sequencing was performed using single-end 100 base pair sequencing.