title: GSM4088772: KKU-213HG; Homo sapiens; RNA-Seq
library_construction_protocol: Confluent cells were trypsinized and preserve in RNAlater® and keep in -80 degree Celcius until use. The extraction was follow the TRIzol® protocol combine with the instruction of RNeasy® kit. Strand-specific, poly-A+ RNA-seq libraries were prepared and whole-transcriptome RNA sequencing (RNA-seq) was performed on the Illumina HiSeq 2000 platform. Briefly, poly-A+ mRNA was purified using Oligo(dT)25 Dynabeads (Life Technologies) followed by fragmentation . The first strand cDNA was synthesized using NEBNext RNA First Strand Synthesis Module (New England BioLabs) and purified using Agencourt RNA Clean XP beads (Beckman Coulter). A dNTP mix containing dUTP was used for the second strand cDNA synthesis, to introduce strand-specificity (NEBNext Ultra Directional RNA Second Strand Synthesis Module, NEB). The resultant cDNA was processed through end-repair and A-tailing, ligated with Illumina PE adapters, and then digested with Uracil-DNA Glycosylase (NEB). The libraries were amplified and barcoded (13 PCR cycles) using the Phusion High-Fidelity PCR Master Mix (NEB). They were purified, pooled in equimolar amounts, and loaded onto a HiSeq flow cell lane. Sequencing runs were performed in paired-end mode (2 x 100-bp reads) following amplification with Illumina's cBot cluster generation system.
attributes.tag: GEO Accession
attributes.value: GSM4088772