RNA-seq of mus musculus MLE12 cells following Six1 Expression
Using the purified RNA, PAC-seq libraries were prepared as previously described (Routh et al. 2017). One g of total RNA was reverse transcribed with the partial P7 adapter (Illumina_4N_21T) and dNTPs with the addition of spiked-in azido-nucleotides (AzVTPs) at 5:1. The p5 adapter (IDT) was Click-ligated to the 5end of the cDNA with CuAAC. The cDNA was then amplified for 16 cycles using the Universal primer and 3 indexing primer, followed by purification on a 2% agarose gel with extraction of the 200-300 base pair amplicon. Barcoded libraries were then pooled and sequenced with single-end, 150 base-pair reads on a Nextseq 550 (Illumina). RNA-seq reads were aligned to the mouse genome (GRCm38, mm10) using Gencode VM23 using bowtie and DeSeq2 was used to detect differential expression