RNAseq of rattus: large4:SFO
RNA extraction and sequencing were performed at the ABM laboratory in Vancouver. Dissected SFO samples from each of the 24 male rats from large litter control litters were pooled into four samples (of six SFO each), and 24 male rats from small litters were pooled into four samples (of six SFO each). RNA extraction was performed using the PureLink RNA Mini Kit (Thermo Fisher Scientific), and analysed using a Bioanalyser (Agilent). RNA integrity number values were assessed using the RNA 6000 pico kit on an Agilent 2100 Bioanalyzer; all RIN values were between 7.7 and 8.6. Two micrograms of sample were prepared for library construction using the TruSeq Standard mRNA Library Prep Kit (Illumina). Briefly, the RNA was subject to polyA enrichment, fragmentation, and cDNA synthesis. Indexed adaptors were ligated to the cDNA library, and then libraries were pooled in equal molar ratio. Quality of library was assessed using an Agilent Bioanalyser prior to sequencing; library sizes ranged from 401 to 427. Sequencing was performed on the NextSeq 500 platform with 2x 75 bp end sequencing. For each sample, sequencing produced between 42.8 and 54.7 million reads: reads were aligned to Rat Rnor 6.0 reference genome and annotation (GCF_000001895.5) using Hisat2 with the parameters (--rf --mm --dta); mapping rates were between 86.76 - 88.52%.