GSM2649273: LY2-NT-shRNA-n-3A; Homo sapiens; RNA-Seq
RNA was extracted using standard BGI extraction protocols. RNA was extracted with average concentrations of 500 ng/µl determined by QubitFluorometer, Agilent 2100 bioanalyser, and nanodrop. BGI analysed the RNA integrity using the Agilent 2100 bioanalyser achieving a >9.5 RNA integrity number for all samples. Total quantity of RNA was above the cut off of ≥ 5µg LIbrary construction was carried out using Illumina®TruSeq  RNA Sample Prep Kit. After the total RNA extraction and DNase I treatment, magnetic beads with Oligo (dT) are used to isolate mRNA(for eukaryotes) or by removing rRNAs from the total RNA (for prokaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. The suitable fragments are selected for the PCR amplification as templates. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library.
GEO Accession
GSM2649273