title: GSM8787417: TIGK 60C-3; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was harvested after 10 minutes in -25°C, 37°C, and 60°C using Trizol and purified with an RNA Clean & Concentrator-25 kit (Zymo, Tustin, CA, USA), and treated with DNAse (ThermoFisher Scientific, Waltham, MA). RNA integrity numbers (RIN) were checked with Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). All samples had an RIN between 8.9-9.4. 1 ug of total RNA was used for construction of sequencing libraries. The library preparation, genome mapping, and raw gene counts for mRNA-sequencing analysis were performed by Novogene (Novogene America). The following methods were those provided by Novogene. Libraries generated for mRNA-sequencing were checked with Qubit and real-time (RT) PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina sequencing platforms (Illumina, Omaha, NE, USA) and clean data was obtained by using in-house Perl scripts from Novogene (Novogene America, Sacramento, CA, USA). Hisat2 (v2.0.5) was used to index and map reads to the human reference genome (GRCh38/hg38), and the mapped reads were assembled by StringTie (v1.3.3b) [22]. Read numbers mapped to each gene were counted in R using the FeatureCounts package (v1.5.0-p3) and an FPKM of each gene was calculated [23].