GSM6523079: B16-F10 cells poly(A) seq D; Mus musculus; RNA-Seq
RNA was harvested using Direct-zol RNA MiniPrep kit (Zymo Research) Poly(A)-seq:.RNA was isolated from cell pellets with the Direct-zol RNA MiniPrep kit (Zymo Research). Protocol was adapted from Derti et al. 2012. In brief, 5 ug of RNA was heated at 65 degrees for 2 minutes and then placed on ice. Poly(A) selected mRNA was isolated using DynaBeads (ThermoFischer 61006) per the manufacturer's instructions. 1st strand cDNA synthesis using SuperScript IV Reverse Transcriptase (ThermoFischer 18090010) and the primer RKB4087 (a modified oligo d(TVN) primer) to generate cDNA reads which begin synthesis just before the poly(A) tail. This mixture was then purified using a 2x SPRI bead clean up and then followed by 2nd strand synthesis and then a 1x SPRI bead clean up. This cDNA library was then subject to PCR (NEBNExt High Fidelity 2x Master Mix) and primers RKB4089 (universal primer) and RKB4090-RKB4101 (indexed primers). The final libraries generate a broad range of fragment sizes, as such we gel extracted libraries from a 2% agarose gel corresponding to 300-500 bp size. Following extraction and purification, library size was analyzed with a 4200 TapeStation System before sequencing. RNA-seq:Poly(A)-selected, unstranded Illumina libraries were prepared following the TruSeq protocol per the manufacturer's instructions. Library size and distribution was analyzed with a 4200 TapeStation System before sequencing.