GSM5017733: Metes_Post_B1; Mus musculus; RNA-Seq
Dissected oviduct samples were collected in duplicate for each of the four stages across both anterior and posterior regions. Oviduct tissue was manually disrupted and the lysate spun through a QIAshredder column (Qiagen, cat#: 79656) to fully homogenize. Total RNA was isolated from each sample by addition of Trizol (Thermo Fisher) and the sample was transferred to a Phasemaker tube (Thermo Fisher). Total RNA was extracted following the protocol supplied by the manufacturer and further cleaned up using a RNeasy MinElute Cleanup Kit (Qiagen). RNA integrity number (RIN) was measured using an Agilent Bioanalyzer and 100 ng of RNA was used for the TagSeq protocol. The fragmentation/RT mix was prepared and added to each RNA sample, then heated to 95°C for 2.5 minutes on a Thermocycler and immediately put on ice for 2 minutes. After cooling and addition of the template switching oligo and SmartScribe RT, the fragmented RNA reaction was incubated at 42°C for 1hr, 65°C for 15 min. Next an AmPure bead clean-up was completed for the cDNA before it was amplified to incorporate the Illumina sequencing primer site, followed by another cleanup. The remaining portions of the Illumina adapter (the i5 and i7 indices) were then added through an additional 4 cycles of PCR. Final libraries were quantified with PicoGreen then pooled equally for size selection using the Blue Pippin from 355-550 bp.