RNA Seq of mouse: TI cre- 3
Illumina-compatible cDNA libraries with RiboZero Gold depletion were constructed according to the TruSeq Stranded Total RNA Reference Guide (Illumina Document #1000000040499v00, TruSeq Stranded Total RNA Gold Illumina Kit #20020598). Final cDNA library concentrations were determined using the Qubit dsDNA BR assay kit (Thermo Fisher Scientific: Guide MAN0002325 MP 32850, Kit #Q32850). The quality of the libraries was evaluated by running each on an Agilent 4150 Tapestation using the DNA 1000 Screentape kit (Agilent: Publication Part Number: G2991-90031, Kit #5067-5583). The electropherogram of each library was analyzed using Agilent 4150 Tapestation Analysis Software (Version 3.1) with a range of 200600 bp to determine the average library size. Average size and concentration were used to calculate the molarity of each library. Libraries were pooled and denatured according to the Illumina Denature and Dilute Libraries Guide for the NextSeq System (Illumina Part #15048776). The denatured libraries were loaded onto an Illumina NextSeq 550 v2.5 Mid 150 output reagent cartridge (Illumina #20024904) at a final concentration of 1.5 pM in HT1 buffer, with 1% PhiX control library v3 (Illumina #FC-110-3001) added. Paired-end 75 bp single-index sequencing was performed, generating approximately 7.5 million paired-end reads per sample.