title: GSM8949919: bulkRNA-seq, HF187440; Homo sapiens; RNA-Seq
library_construction_protocol: Bulk RNA-seq: The tissues were disrupted in Agencourt RNAdvance tissue lysis buffer (Beckman Coulter, Brea, CA) using a tissue ruptor (Omni International, Kennesaw, GA, # 19-040E). Total RNA was extracted in a Biomek FXp automation workstation according to the manufacturer's instructions for tissue-specific extraction. Bulk RNA-seq: Universal Plus mRNA-Seq with NuQuant Human Globin AnyDeplete kit from Tecan (# 0521-A01) was used for the generation of RNA-seq libraries derived from poly(A)-selected RNA according to the manufacturer's instructions. Universal Plus mRNA-Seq libraries contain dual (i7 and i5) 8 bp barcodes and an 8 bp unique molecular identifier (UMI), which enable deep multiplexing of NGS sequencing samples and accurate quantification of PCR duplication levels. Approximately 300ng of total RNA was used to generate the libraries. The Universal Plus mRNA-Seq workflow consists of poly(A) RNA selection, RNA fragmentation, and double-stranded cDNA generation using a mixture of random and oligo(dT) priming, end repair to generate blunt ends, adaptor ligation, strand selection, AnyDeplete workflow to remove unwanted ribosomal and globin transcripts, and PCR amplification to enrich final library species. All library preparations were performed using a Biomek i7 laboratory automation system (Beckman Coulter). Normalized pooled libraries were sequenced on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) to a target depth of 35 million read pairs (70 million paired-end reads) per sample using a paired-end 100 base pair run configuration. To capture the 8-base UMIs, libraries were sequenced using 16 cycles for the i7 index read and 8 cycles for the i5 index read.