title: GSM8196059: AidWT_RemSD_b34_8; Mus musculus; RNA-Seq
library_construction_protocol: Library preparation and sequencing for TagSeq, a form of 3' RNA sequencing were performed by the Genomic Sequencing and Analysis Facility (GSAF) at The University of Texas at Austin. Total RNA was isolated from each sample (0.375 mL) by addition of Trizol (1.125 mL; Thermo Fisher) and the sample (1.4 mL) 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 Tag-Seq protocol. The fragmentation/RT mix was prepared then added to each RNA sample and then heated to 95 degrees for 2.5 minutes on a Thermocyler then 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 42°C for 1hr, 65°C for 15 min. Next an AmPure bead clean-up was completed for the cDNA before it was amplified for 18 cycles, also incorporating the Illumina sequencing primer site, followed by another cleanup. The remaining portions of the Illumina adapater (the i5 and i7 indices) were then incorporated 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.