title: GSM6051782: W1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA cellular RNA was extracted (RNeasy Mini Kits, Qiagen) and quantified using Agilent 4200 TapeStation (Agilent Technologies). Total RNA libraries were generated using the Illumina TruSeq Stranded Total RNA Sample Preparation Guide, Revision E. The first step involved the removal of ribosomal and mitochondrial RNA using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. Following purification, remaining RNA was fragmented using divalent cations under elevated temperature, which were then copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Subsequently, a single adenosine base was added to each of the cDNA fragments, followed by ligation of an adapter. The products were purified and enriched with PCR to create the final cDNA library. The cDNA libraries were validated using KAPA Biosystems primer premix kit with Illumina-compatible DNA primers and Qubit 2.0 fluorimeter. Quality was examined using an Agilent Bioanalyzer Tapestation 2200. The cDNA libraries were pooled at a final concentration 1.8pM. Cluster generation and 100 bp paired-read dual-indexed sequencing was performed on Illumina NextSeq 500 (Children's Hospital of Pittsburgh, University of Pittsburgh).