RNA-Seq MDA-MB-468 MeCP2
RNA quality was determined using RNA Screen Tape (Agilent). Ribosomal RNA depletion was achieved using NEB Next rRNA Depletion Kit (Human/Mouse/Rat) (NEB # E6310X ). RNA fragmentation, double stranded cDNA and adaptor ligation was generated using NEBNext Ultra II Directional RNA Library Prep according to the manufacture protocol (NEB # E7760L). PCR enriched libraries were quantified by Qubit and equimolar indexed libraries (different samples had different indexes for multiplexing) were pooled. Pooled libraries were quantitatively checked using the Agilent Tapestation 2200 and quantified using Qubit. The libraries were then diluted to 200 pM and spiked with 2% phiX libraries (Illumina control). The transcriptome sequencing was performed on the barcoded stranded RNA-Seq libraries using Illumina NovaSeq 6000 SP flow cell, paired-end reads (2  50 bp).  The RNA-Seq reads were normalized by RPKM and assembled by mapping reads directly to the annotated human reference genome using the DNASTAR SeqMan software (DNASTAR, Inc., Madison, WI).  Differential gene expression levels were quantified using Fishers Exact Test Signal Search in the DNASTAR ArrayStar software package (DNASTAR, Inc., Madison, WI). Differentially expressed genes were filtered if they met the criterion for a two-fold change, a P-value that was less than .05 at a 95 % confidence interval. For each comparison, genes were sorted based on fold change, from low to high.  The results were ported into Excel spreadsheets where the log2 of the fold change for each gene was calculated.