title: GSM3984151: 41; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated using the standard TRIzol protocol and re-suspended in 85 uL Ultrapure Distilled water (Invitrogen). DNase treatment was performed in solution with the RNase-Free DNase Set (Qiagen) using 5 uL DNase I and 10 uL RDD buffer, followed by a column cleanup using the RNeasy Mini Kit (Qiagen) with two additional washes (75% ethanol) before elution Libraries were prepared by the Van Andel Genomics Core from 500 ng of total RNA using the KAPA RNA HyperPrep Kit with RiboseErase (v1.16) (Kapa Biosystems). RNA was sheared to an average of 300 bp. Prior to PCR amplification, cDNA fragments were ligated to Bioo Scientific NEXTflex Adapters (Bioo Scientific). Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies Inc.) and QuantiFluor® dsDNA System (Promega Corp.). Individually indexed libraries were pooled and 75 bp, single end sequencing was performed on an Illumina NextSeq 500 sequencer using a 75 bp HO sequencing kit (v2) (Illumina Inc.), with all libraries run across 4 flowcells to return a minimum read depth of 40M read pairs per library.  Base calling was done by Illumina NextSeq Control Software (NCS) v2.0 and output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.