GSM9740242: Mouse liver tissue 24h after injection of saline, rep5; Mus musculus; RNA-Seq
Liver tissue samples (~25-35mg) were homogenised in 500uL of NucleoZOL reagent (Macherey-Nagel) using a glass dounce homogeniser. Water was added and samples centrifuged to remove debris and contaminants according to the manufacturer's directions. Crude extracts were mixed 1:1 with 100% ethanol and RNA captured on a Zymo-Spin IC column (Zymo Research). Columns were washed, DNase I digestion was performed on-column, then columns were washed again and RNA eluted according to the manufacturer's directions. RNA integrity was verified using the Agilent TapeStation platform and only samples with a RIN above 7.5 were progressed for sequencing (typical RIN values were around 9). Sequencing libraries were prepared by Azenta Life Sciences using the VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme). Poly(A) mRNA was isolated using VAHTS mRNA Capture Beads. mRNA was fragmented and primed using 2× Frag/Prime Buffer, and first-strand cDNA was synthesised using 1st Strand Buffer 3/1st Strand Enzyme Mix 3. Second-strand cDNA synthesis was performed using 2nd Strand Buffer 2/2nd Strand Enzyme Super Mix 2 (dUTP method), producing strand-specific libraries. Following end repair, dA-tailing, and T-A ligation of adaptors, fragments of approximately 260–360 bp (insert size 200–300 bp) were size-selected using VAHTS DNA Clean Beads. Libraries were amplified by PCR for 12–15 cycles using P5 and P7 primers, cleaned up with VAHTS DNA Clean Beads, validated on an Agilent 4200 Bioanalyzer, and quantified by Qubit 3.0 Fluorometer. Libraries were multiplexed and sequenced on an Illumina NovaSeq 6000 using a 2×150 paired-end configuration.