GSM8636242: K20200104,Adelaide,Testis; Phascolarctos cinereus; RNA-Seq
Total RNA samples were treated with Turbo DNase (Invitrogen) and RNA cleanup was done following manufacturer's instructions using the RNeasy Mini Kit (Qiagen). Strand-specific RNA sequencing libraries for were prepared as described in (Zhang Z, Theurkauf WE, Weng Z, Zamore PD. Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection. Silence. 2012;3(1):9. Published 2012 Dec 28. doi:10.1186/1758-907X-3-90).  In brief, total RNA was isolated from frozen koala tissue samples via the mirVana miRNA Isolation kit and then rRNA depleted using the RiboZero™ Gold rRNA Removal Kit (Human/Mouse/Rat) (Illumina). RNAs greater than 200nt were selectively recovered using the RNA Clean & Concentrator-5 kit(Zymo Research).  RNA samples were fragmented and reverse transcribed. dUTP was incorportated during second strand synthesis for strand specificity. End repair and A-tailing was performed followed by adapter ligation and uracil-DNA glycosylase (UDG) treatment.  ERCC spike-ins and UMI tags are added to the libraries. Finally, the library was PCR amplified.