title: GSM2209021: mock_04; Homo sapiens; RNA-Seq
library_construction_protocol: The cells were harvested with TRIzol (Life Technologies) at either day 2 or day 3 for RNA. RNA-Seq libraries were prepared using Illumina's TruSeq mRNA Library Prep kit according to manufacturers LT (low-throughput) protocol. Briefly, mRNA was isolated from total template RNA using oligo-dT beads, first strand cDNA was synthesized using kit provided actinomycin-D mix with added SuperScript III reverse transcriptase (Invitrogen), followed by second strand synthesis using a marking mix that incorporates dUTP in place of dTTP. 3' ends of ds-cDNA were adenylated with a single 'A' nucleotide to serve as a complementary overhang for ligation of the single 'T' of indexed Illumina sequence adapters. Adapter ligated fragments were size selected using a double-sided SPRI bead clean-up (Ampure-XP, Beckman Coulter) to target fragments in the ~300 base pair range. Purified, size selected library fragments then underwent 15 cycles of PCR amplification before a final SPRI bead clean-up to remove primer dimers. Library fragments were sized using the DNA 1000 protocol for the BioAnalyzer 2100, and final library quantification was performed using the Kapa Library Quantification qPCR Kit for Illumina (Kapa Biosystems). Libraries were then pooled, and multiplex sequencing was performed on the Illumina NextSeq 500 using a 300 cycle high output kit according to manufacturers instructions. A total of two high output runs were performed using the same multiplexed library pool in order to achieve the desired quantity of reads per sample.
attributes.tag: GEO Accession
attributes.value: GSM2209021