alias: 19_5dKO2.fq.gz
title: RNA-seq of the skin of mus musculus: P1(E19.5) Cdc42 KO mouse-2
design: Total RNA was extracted from the skin of wild-type (WT) and Cdc42 KO mice at E17.5 and P1 (n = 2 per genotype for each time point) using TRIzol following the manufacturer s instructions (Invitrogen). An additional DNase I digestion was performed to remove genomic DNA contamination. RNA quality and purity were checked using the NanoDrop (Thermo Fisher Scientific, Inc., USA).The total RNA samples are first treated with DNase I to degrade any possible DNA contamination. Then the mRNA is enriched by using the oligo(dT) magnetic beads (for eukaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments (about 200 bp). Then the first strand of cDNA is synthesized by using random hexamer-primer. Buffer, dNTPs, RNase H and DNA polymerase I are added to synthesize the second strand. The double strand cDNA is purified with magnetic beads. End reparation and 3'-end single nucleotide A (adenine) addition is then performed. Finally, sequencing adaptors are ligated to the fragments. The fragments are enriched by PCR amplification. During the QC step, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used to qualify and quantify of the sample library. The library products are ready for sequencing via Illumina HiSeqTM 2000.
identifiers.namespace: Southern Medical University
identifiers.id: 19_5dKO2.fq.gz