RNA sequencing data of human hepatocyte
Commercially available human hepatocyte RNA was used for RNA sequencing. The integrity of the RNA was analysed by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The library preparation for RNA sequencing was performed using TruSeq RNA Sample Prep Kit v2 (Illumina Inc., San Diego, CA, USA) according to the protocol outlined in “TruSeq RNA Sample Preparation Guide”. Briefly, the total RNA in RNA Stable tube was recovered by adding 50μl of sterile water provided with the kit and hydrated for 20 minutes at room temperature until the pellet was completely dissolved. 50μl (~1.5-3.5μg) of total RNA was subjected to Poly A purification of RNA. Purified RNA was fragmented for 4 minutes at elevated temperature (94 degree C) in the presence of divalent cations and reverse transcribed with Superscript III Reverse transcriptase by priming with Random Hexamers. Second strand cDNA was synthesized in the presence of DNA polymerase I and RNaseH. The cDNA was cleaned up using Agencourt Ampure XP SPRI beads (Beckman Coulter, Brea, CA, USA). Illumina Adapters were ligated to the cDNA molecules after end repair and addition of A base. SPRI cleanup was performed after ligation. The library was amplified using 13 cycles of PCR for enrichment of adapter ligated fragments. The prepared library was quantified using Nanodrop and validated for quality by running an aliquot on High Sensitivity Bioanalyzer Chip (Agilent Technologies, Santa Clara, CA, USA). TruSeq Cluster Kit v3 (cBot - HS) (Illumina Inc., San Diego, CA, USA) with TruSeq v3 Flow Cell (Illumina Inc., San Diego, CA, USA) was used to sequence the transcriptome library on Hiseq 2000 platform (Illumina Inc., San Diego, CA, USA).