GSM5379819: WT2_S12; Mus musculus; RNA-Seq
Six-week old mice were used to isolate mouse skin for RNA extraction for subsequent RNA-Seq.  Upon sacrifice, the dorsal skin was shaved, the mice immediately placed on ice and a rectangular piece of skin was surgically removed using ice-cold micro-dissecting scissors and forceps. The skin was divided into 4 strips of 3-5mm each, immediately snap frozen in liquid nitrogen and stored at -80C until further processing. All mouse skin isolation was performed within 60-70 seconds of sacrifice to prevent RNA degradation. Samples were cut and placed in lysis bead tubes containing RLT buffer supplemented with DTT. The samples were homogenized using the Magna Lyser and RNA was harvested using the RNeasy Mini kit (Qiagen). KAPA Stranded RNA-Seq Kit with RiboErase Kit was used with 1 ug of total RNA for the construction of sequencing libraries. Libraries for RNA-Seq were prepared with KAPA Stranded RNA-Seq Kit with RiboErase Kit. The workflow consists of depletion of rRNA by hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase and RNA fragmentaion. First strand cDNA synthesis using random priming followed by second strand synthesis converting cDNA:RNA hybrid to double-stranded cDNA (dscDNA), and incorporates dUTP into the second cDNA strand.  cDNA generation is followed by A-tailing, adaptor ligation and PCR amplification. Different adaptors were used for multiplexing samples in one lane.