title: GSM3137676: Hepatocytes Rep 1; Mus musculus; RNA-Seq
library_construction_protocol: Two replicates were used for each stage. Total RNA was isolated from 3*104 cell aliquots using RNeasy Micro Kit (Qiagen, ID:74004) following manufacturers' instruction. Total RNA was resuspended in 50 ul BTE solution (10 mM Bis-tris pH6.7, 1 mM EDTA). RNA secondary structures were removed by heating the samples at 65 C for 5 min, followed by incubation on ice. Oligo-dT25 dynabeads (Invitrogen #610-02) were used for selecting polyA mRNA. Beads were washed 3 times with 2X Oligo-dT binding buffer (OBB: 20 mM Tris ph7.5, 1 M LiCl, 2 mM EDTA), resuspended in 50 ul 2X OBB and added to RNA. Samples were incubated 5-10 min at RT while gently shaking. Samples were transferred to a  (Thermo Fisher Scientific, #12321D) rack, and beads were allowed to separate for 3-5 min. Beads were washed 3 times with 200 ul Oligo-dT washing buffer (OWB: 10 mM Tris Ph7.5, 150 mM LiCl, 1 mM EDTA) and polyA mRNA was eluted into 10 ul BTE. Samples were incubated 2 min at 80C and beads were allowed to separate on the magnetic rack (Thermo Fisher Scientific, #12321D). The solution was transferred to a fresh tube. Chemical RNA fragmentation was achieved using Mg2+. On ice, for each 10 ul sample volume, 4 ul 5X 1st strand buffer from SuperScript III RT kit (Invitrogen #18080-044) and 2 ul 50 uM random hexamers (Roche) were added. Samples were incubated 16 min at 94C in PCR machine and ramped down to 4C. First strand synthesis was performed using SuperScript III (Invitrogen #18080-044), following manufacturers'' instructions and adding 1ug actinomycin D (Sigma #A1410) per reaction. Samples were incubated in PCR machine according to the following scheme: 10 min at 25C, 20 min at 42C, 30 min at 50C, 10 min at 55C. Samples were kept on ice. First strand cDNA was purified using conventional phenol:chloroform:isoamyl extraction. cDNA was precipitated using 3 M NaAc, 1.ul 20mg/ml glycogen (Roche, #10901393001) and  1 ml 100% EtOH. Samples were resuspended in milliQ water. Second strand synthesis incorporating dUTP was performed as following: for each sample, 2 ul First Strand bugger (from SuperScript III kit; Invitrogen #18080-044), 0.5 ul 0.1 M DTT, 5 ul 10X second strand buffer (dNTPs free; NEBnext #B6117S), 2 ul 10 mM dNTPs (with dUPT substituting in place of dTTP), 2 ul DNApol I (20 U; NEB #M0209), 0.5 ul RNAseH (2.5 U: NEB #M0297) were added. Samples were incubated 2-3h at 16C. cDNA was purified using MiniElute kit (Qiagen, #28004) and samples were eluted in 10ul RNase-free water. Libraries were generated using ThruPLEX DNA-seq 48S Kit (Rubicon Genomics, #R400427). Libraries were quantified by qPCR using the NEBNext Library quantification kit for Illumina (NEB, #E7630L). Libraries were diluted to 8 nM and pooled for multiplexing. The diluted concentrations and were quantified a second time using NEBNext kit. Libraries were denatured in 0.2 M NaOH and loaded into NextSeq 75-cycles High Output v2 Kit (Illumina, FC-404-2005) cartridge at a concentration of 2.5-3 pM. Sequencing runs were performed in an Illumina NextSeq 500 machine
attributes.tag: GEO Accession
attributes.value: GSM3137676