title: GSM8598563: Liver, Paracetamol injury, hAAM, rep 2; Mus musculus; RNA-Seq
library_construction_protocol: Approximately 10 mg frozen liver tissue was homogenized in 900 µL Qiazol lysis reagent (Qiagen) using the tissue tearor. Chloroform was mixed and incubated (5 min) before centrifugation (12000 g, 15 min, 4 °C). Aqueous phase was separated and RNA extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. 14 Libraries were prepared from total-RNA samples using the NEBNext Ultra 2 Directional RNA library prep kit for Illumina (#E7760) and the NEBNext rRNA Depletion kit (Human/Mouse/Rat) (#E6310) according to the provided protocol. 500ng of total-RNA was added to the ribosomal RNA (rRNA) depletion reaction using the NEBNext rRNA depletion kit (Human/mouse/rat). This step uses specific probes that bind to the rRNA in order to cleave it. rRNA-depleted RNA was then DNase treated and purified using Agencourt RNAClean XP beads (Beckman Coulter Inc, #66514). RNA was then fragmented using random primers before undergoing first strand and second strand synthesis to create cDNA. Fragmentation time was dependent upon the quality of each total RNA sample. cDNA was end-repaired before ligation of sequencing adapters, and adapter-ligated libraries were enriched by 10 cycles of PCR using NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (NEB, #E6440 or E6442).