GSM3178874: Cre neg_1; Mus musculus; RNA-Seq
Bladder urothelium separated from stroma by trypsin digestion. Dissected tissues were placed directly into RLT Plus lysis buffer (Qiagen) for later processing. For each batch of harvest, dissected tissues from multiple embryos were pooled into one lysate. Total RNA was purified from the RLT Plus lysates using the RNeasy Plus Micro Kit (Qiagen) according to manufacturer's instructions. The following is an excerpt from Hou et al, Sci Rep. 2015; 5:9570, the manuscript that details the protocol employed. Poly-A-tailed mRNA is isolated from total RNA using oligo-dT beads. Purified mRNA is then fragmented with heat in fragmentation buffer. First strand cDNA is then synthesized using random hexamer oligos containing partial Illumina 3′ adaptor sequence. After RNA removal, a modified oligo containing partial Illumina's 5′ adaptor is then ligated to the 5′ of the single stranded cDNA. The library is then amplified by PCR using oligos that contain full Illumina adaptor sequences and our in-house index sequences.