title: GSM4873921: 31; Mus musculus; RNA-Seq
library_construction_protocol: Brain endothelial cells were cultured as biological replicates in 12-well plates (i.e. 1 brain per well) and grown to confluence. Total RNA was extracted using miRNeasy Mini Kit (Qiagen, 217004) and used for sequencing library construction. RNA samples were quantified using NanoDrop™ One Spectrophotometer (Thermo Scientific) and analyzed for integrity using 4200 TapeStation (Agilent). Levels of remaining DNA were checked using Qubit fluorometer (Invitrogen). DNA amounts did not exceed 10% of the total amount of NA Sequencing libraries for Illumina sequencing were prepared using 100ng of total RNA per sample. Library prep was carried with the Universal Plus mRNA-Seq kit (NuGen, 0520-A01), as written in the product manual (NuGen, M01485 v5). In brief, RNA underwent poly-A selection, enzymatic fragmentation, and generation of double-stranded cDNA using a mixture of oligo (dT) and random priming. The cDNA underwent end repair, ligation of dual-index adaptors, strand selection, and 17 cycles of PCR amplification. The number of cycles was determined by qPCR on a small aliquot of the un-amplified libraries. All intermediate purification steps, and final library purification was carried out using Agencourt AMPure XP Beads (Beckman Coulter A63881). Final amplified libraries were measured with the Qubit 1X dsDNA HS Assay Kit (Invitrogen, Q33231), and fragment size distribution was confirmed using the D5000 ScreenTape assay (Agilent, 5067-5588, 5067-5589).  Concentration of the final library pool was confirmed by qPCR, and subjected to test sequencing in order to check sequencing efficiencies and adjust accordingly proportions of individual libraries. The pool was purified with the Agencourt AMPure XP Beads (Beckman Coulter A63881), quantified by qPCR using KAPA Library Quantification Kit and run on a NovaSeq6000 SP flow cell, 2x50 nt, two lanes of , at the University of Illinois Roy J. Carver Biotechnology Center High-Throughput Sequencing and Genotyping Unit.
attributes.tag: GEO Accession
attributes.value: GSM4873921