title: GSM4162307: TW_2_1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA in tissue was harvested using Trizol reagent (Life Technologies) according to manufacturer instructions. The purified mRNAs were used to construct the cDNA library with the NEB RNA library construct kit. The quality-ensured libraries were used for pair-end deep sequencing on Illumina HiSeq Sequencer. A total of 1.5 μg RNA per sample was used as input material for RNA sample preparations. Sequencing libraries were generated using a NEBNext UltraTM RNA library prep kit Illumina (New England Biolabs, Ipswich, MA, USA) according to manufacturer instructions, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, and fragmentation was performed using divalent cations under an elevated temperature in NEBNext First Strand Synthesis reaction buffer (5×; New England Biolabs). First-strand cDNA was synthesized using a random hexamer primer and M-MuLV reverse transcriptase (RNase H−; New England Biolabs). Second-strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3′ ends of the DNA fragments, NEBNext Adaptor (New England Biolabs) with a hairpin loop structure was ligated to prepare for hybridization. To select cDNA fragments of the correct length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, MA, USA), after which 3 µL USER enzyme (New England Biolabs) was incubated with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by 5 min at 95°C. PCR was performed using a Phusion high-fidelity DNA polymerase, universal PCR primers, and an Index (X) primer (New England Biolabs). Products were purified (AMPure XP; Beckman Coulter), and library quality was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies).
attributes.tag: GEO Accession
attributes.value: GSM4162307