GSM9428722: Hippocampus of Mrpl40 +/+,Slc25a1 +/+, biol rep 2; Mus musculus; RNA-Seq
RNA was isolated from samples using Qiazol phase separation followed by cleanup with RNeasy 96 Kit per manufacturer's instruction. Isolated RNA sample quality was assessed by both RNA Tapestation assay and High Sensitivity RNA Tapestation assay (Agilent Technologies Inc., California, USA and quantified by Infinite F Nano+ 200 Pro Tecan® (Tecan, Switzerland) Paramagnetic beads coupled with oligo d(T)25 are combined with total RNA to isolate poly(A)+ transcripts based on NEBNext® Poly(A) mRNA Magnetic Isolation Module manual (New England BioLabs Inc., Massachusetts, USA). Prior to first strand synthesis, samples are randomly primed (5´ d(N6) 3´ [N=A,C,G,T]) and fragmented based on manufacturer's recommendations. The first strand is synthesized with the Protoscript II Reverse Transcriptase with a longer extension period, approximately 30 minutes at 42⁰C. All remaining steps for library construction were used according to the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England BioLabs Inc., Massachusetts, USA). Final libraries quantity was assessed by Qubit 2.0 (ThermoFisher, Massachusetts, USA) and quality was assessed by TapeStation HSD1000 ScreenTape (Agilent Technologies Inc., California, USA). Final library size was about 450bp with an insert size of about 300bp. Illumina® 8-nt dual-indices were used. Equimolar pooling of libraries was performed based on QC values and sequenced on an Illumina® NovaSeq X Plus (Illumina, California, USA) with a read length configuration of 150 PE for 60M PE reads per sample (30M in each direction).