Rna 600 nano chips

Grey matter tissue was dissected from the cortical ribbon of the Brodmann Area 9 gyral crest. Total RNA was extracted using the Promega Maxwell RSC simplyRNA Tissue Kit (Cat No. AS1340) according to the manufacturer’s protocol. The integrity and quality of RNA were verified by an Agilent 2100 Bioanalyzer using RNA 600 Nano Chips (Cat No. 5067-1511). Only cases with a RNA Integrity Number (RIN) of 4 or higher using an Agilent Bioanalyzer instrument were selected for study. Paired end poly-A selected mRNA sequencing libraries with a targeted library size of 80M reads were generated using a Kapa Stranded mRNA-Seq Kit according to the manufacturer instructions and sequenced on an Illumina MiSeq instrument by the Boston University Microarray and Sequencing Core. Samples were sequenced in four batches due to study size and were distributed among batches as to avoid any confounding of status, age at death, RIN, or other important experimental variables.

Total RNA was extracted from approximately 10,000 synchronized L4 animals using the standard Trizol, chloroform, isopropanol protocol with on-column DNAse digest. Sample quality and quantity were assessed on the Agilent Bioanalyzer using RNA 600 Nano chips, and only samples with an RIN score ≥9 were used for library construction. mRNA was enriched from 1 μg of total RNA using NEXTflex Poly(A) Beads (Bioo Scientific, Austin, TX, NOVA-512979), and strand-specific directional libraries were generated using the NEXTflex Rapid Directional RNA-Seq Kit (Bioo Scientific). For each genotype, samples were prepared from 3 biological replicates and indexed with distinct barcodes. Quality and fragment size distribution of synthesized cDNA libraries were assessed on the Agilent Bioanalyzer using DNA 1000 chips. Library concentrations were quantified using the NEBNext Library Quant Kit (New Englad Biolabs, Ipswich, MA, E7630S), and each library was normalized to 10 nM in TE buffer prior to pooling. Multiplexed libraries were sequenced using 100-bp paired-end reads on the Illumina HiSeq 4000 platform at the UCSF Center for Advanced Technologies.