GSM3581229: Cont_5; Mus musculus; RNA-Seq
Telogen dorsal skin was shaved and a ~4 x 4 cm region of skin was collected and incubated overnight in 0.25% trypsin at 4 degrees Celsius.  The next day, the epidermis were scraped from the dermis using a razor blade, minced in trypsin, and passed through a 100 um nylon mesh to remove hair shafts.  The cells were lysed in 350 uL RLT buffer per 5 x 10^6 cells, and processed using RNeasy Mini Kit (Qiagen).  1.5 ug of total RNA with RIN > 7 was submitted to Novogene Corporation for RNA-Seq, and ~20 x 106 reads were collected per sample. After QC procedures, mRNA was enriched using oligo(dT) beads. Next, the mRNA was fragmented randomly by adding fragmentation buffer, then cDNA was synthesized by using mRNA template and random hexamers, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I were added to initiate second-strand synthesis. After a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library was completed through size selection and PCR enrichment. The quality control of the library consisted of three steps:  (1) Qubit 2.0: tested the library concentration preliminarily.  (2) Agilent 2100: tested the insert size.  (3) Q-PCR: quantified the library effective concentration precisely. The qualified libraries were fed into HiSeq/MiSeq sequencers after pooling according to its effective concentration and expected data volume.