title: GSM5252679: 633_BRQ_2h_rep2; Mus musculus; RNA-Seq
library_construction_protocol: Qiagen RNEasy PLUS kit with additional on-column DNAse step. Total RNA is quantified using the Quant-iT™ RiboGreen® RNA Assay Kit and normalized to 5ng/ul. An aliquot of 200ng for each sample is transferred into library preparation which is an automated variant of the Illumina TruSeq™ Stranded mRNA Sample Preparation Kit. This method preserves strand orientation of the RNA transcript. It uses oligo dT beads to select mRNA from the total RNA sample. It is followed by heat fragmentation and cDNA synthesis from the RNA template. The resultant 500bp cDNA then goes through library preparation (end repair, base 'A' addition, adapter ligation, and enrichment) using Broad designed indexed adapters substituted in for multiplexing. After enrichment the libraries were quantified with qPCR using the KAPA Library Quantification Kit for Illumina Sequencing Platforms and then pooled equimolarly. The entire process is in 96-well format and all pipetting is done by either Agilent Bravo or Hamilton Starlet. Pooled libraries are normalized to 2nM and denatured using 0.1 N NaOH prior to sequencing. Flowcell cluster amplification and sequencing were performed according to the manufacturer's protocols using either the HiSeq 2000 or HiSeq 2500. Each run is a 101bp paired-end with an eight-base index barcode read. Data is analyzed using the Broad Picard Pipeline which includes de-multiplexing and data aggregation. An aliquot of 200ng for each sample is transferred into library preparation which is an automated variant of the Illumina TruSeq™ Stranded mRNA Sample Preparation Kit. This method preserves strand orientation of the RNA transcript. It uses oligo dT beads to select mRNA from the total RNA sample. It is followed by heat fragmentation and cDNA synthesis from the RNA template. The resultant 500bp cDNA then goes through library preparation (end repair, base 'A' addition, adapter ligation, and enrichment) using Broad designed indexed adapters substituted in for multiplexing. After enrichment the libraries were quantified with qPCR using the KAPA Library Quantification Kit for Illumina Sequencing Platforms and then pooled equimolarly. The entire process is in 96-well format and all pipetting is done by either Agilent Bravo or Hamilton Starlet. Pooled libraries are normalized to 2nM and denatured using 0.1 N NaOH prior to sequencing. Flowcell cluster amplification and sequencing were performed according to the manufacturer's protocols using either the HiSeq 2000 or HiSeq 2500. Each run is a 101bp paired-end with an eight-base index barcode read. Data is analyzed using the Broad Picard Pipeline which includes de-multiplexing and data aggregation.
attributes.tag: GEO Accession
attributes.value: GSM5252679