Hiseq 3000 4000 sr cluster kit reagents

Ch25h^fl/fl and Ch25h^ECKO female mice were injected with tamoxifen. Nine brains per genotype were pooled and primary brain microvascular endothelial cells (pMBMEC) were isolated and plated in a 96‐well plate (2 wells/brain). Confluent pMBMEC were left unstimulated or stimulated IL‐1β for 24 h. RNA of three wells was pooled to obtain one replicate for RNA sequencing. The Lausanne Genomic Technologies Facility performed the RNA‐seq. RNA quality was assessed on a Fragment Analyzer (Agilent Technologies), and all RNAs had a RQN between 8.7 and 10. RNA‐seq libraries were prepared from 500 ng of total RNA with the Illumina TruSeq Stranded mRNA reagents (Illumina) using a unique dual indexing strategy, and following the official protocol automated on a Sciclone liquid handling robot (PerkinElmer). Libraries were quantified by a fluorometric method (QubIT, Life Technologies) and their quality assessed on a Fragment Analyzer (Agilent Technologies).
Cluster generation was performed with 2 nM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 SR Cluster Kit reagents and sequenced on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents for 150 cycles (single end). Sequencing data were demultiplexed using the bcl2fastq2 Conversion Software (version 2.20, Illumina).

RNA from frozen half-kidneys of 72 mice were extracted and purified using RNAeasy MiniElute Spin Column (Qiagen). RNA quality was assessed on a Fragment Analyzer (Agilent Technologies). All RNAs had an RNA quality number (RQN) between 7.5 and 9.7. RNA-Seq libraries were prepared from 200 ng of total RNA with the Illumina TruSeq Stranded mRNA reagents (Illumina) using a unique dual indexing strategy and following the official protocol automated on a Sciclone liquid handling robot (PerkinElmer). Libraries were quantified by a fluorometric method (QubIT, Life Technologies) and their quality assessed on a Fragment Analyzer (Agilent Technologies). Clusters were generated with 2 nM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 SR Cluster Kit reagents. Sequencing was performed on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents for 150 cycles (single read). Sequencing data were demultiplexed, filtered for failed reads, and written to FASTQ files using the bcl2fastq2 conversion software (version 2.20, Illumina). Details for RNA-Seq reads mapping are described in Supplemental Methods.