title: GSM8009838: Liver Control_3; Mus musculus; RNA-Seq
library_construction_protocol: Samples of frozen tissue were cut and weighed to obtain  25mg. The tissue was placed into a 1.7mL microfuge tube with 500uL Trizol (Life Technologies) and kept on ice. The tissue was homogenized with a disperser (IKA) with three cycles of 20 seconds of dispersing (speed 6- liver; speed 3- brain) followed by 10 seconds of rest. The tube was kept in an ice water bath during the homogenization. After the homogenization, the tube was centrifuged for 12,000 x g for 1 minute. The supernatant was removed to a new 1.7mL microfuge tube without disturbing cell debris that may have been pelleted. The remainder of the protocol followed the instructions for the Zymo Research Direct-zol RNA miniprep kit with some minor edits. An equal volume of ethanol (90-100%) was added to the supernatant and briefly vortexed. The mixture was transferred to the Zymo Spin IICR column in a collection tube and centrifuged for 30 seconds at 12,000 x g. The flow-through was discarded and 400uL of Direct-zol RNA Wash Buffer was added to the column. The column was centrifuged for 30 seconds at 12,000 x g. A mixture of DNase digestion buffer (75uL) and DNase I (5uL) was added to the column and incubated at room temperature for 15 minutes. After the DNase I incubation, 400uL of Direct-zol RNA PreWash was added to the column, followed by centrifugation for 30 seconds at 12,000 x g. The flow-through was discarded and the PreWash step was repeated. The flow through was discarded and 700uL of Direct-zol RNA Wash Buffer was added to the column and centrifuged for 2 minutes at 12,000 x g. The spin column was removed to a fresh collection tube and centrifuged again for 1 minute at 12,000 x g. To elute the RNA, 30uL of DNase/RNase-free water was added directly to the column and incubated for 5 minutes at room temperature. The RNA was eluted by centrifugation for 1 min at 16,000 x g into a new 1.7mL lo-bind RNase-free microtube. The RNA was stored at -80oC. Library construction is performed according to the Illumina TruSeq mRNA stranded protocol. The input quantity for total RNA will be ~ 550ng and mRNA will be enriched using oligo dT magnetic beads. The enriched mRNA is chemically fragmented for three minutes. First-strand synthesis uses random primers and reverse transcriptase to make cDNA. After second-strand synthesis, the ds cDNA is cleaned using AMPure XP beads, and the cDNA is end-repaired and then the 3' ends are adenylated. Illumina unique dual indexed adapters are ligated on the ends and the adapter ligated fragments are enriched by nine cycles of PCR. The resulting libraries will be validated by qPCR and sized by Agilent Bioanalyzer DNA high-sensitivity chip. The concentrations for the libraries will be normalized and multiplexed together. The multiplexed libraries will be sequenced using paired-end 100 cycles chemistry on the NovaSeq 6000. We will target 20M reads per sample.