HEK293T cells were co-transfected with 3 ug total plasmid in 6-well
plates. After three days, cells were washed twice with PBS and 350 uL of 1:10
mixture of β-mercaptoethanol and Buffer RLT (Qiagen) was added to each
well. While on ice, cells were lysed NA was quantified using a Nanodrop
instrument, and RNA quality was assessed using an Agilent TapeStation 2200 with
RNA ScreenTape (Agilent). Using 1 ug of total RNA input, stranded mRNA sample
preparation was performed with the Illumina TruSeq Stranded mRNA Library Prep
Kit (Illumina) following manufacturer’s protocol except the enzymatic
fragmentation time was reduced from 8 min to 1 min. Libraries were sequenced at
the Duke GCB Sequencing Core as 51 cycles paired-end runs (51PE), in an Illumina
HiSeq 4000 platform. Reads were aligned against the human reference genome
GRCh38 using the aligner STAR v2.4.1a45 following the proposed 2-pass strategy to first
identify a splice junction database to improve the overall mapping quality. Gene
counts were estimated with featureCounts from the subread package
v1.4.6-p646 , using
gene annotations from Refseq47 and allowing for multimapping reads (-M and --fraction arguments). Differential
expression analyses were performed using the DESeq2 package48 filtering out non-expressed genes and
fitting a negative binomial generalized linear model to find significantly
differentially expressed genes.
plates. After three days, cells were washed twice with PBS and 350 uL of 1:10
mixture of β-mercaptoethanol and Buffer RLT (Qiagen) was added to each
well. While on ice, cells were lysed NA was quantified using a Nanodrop
instrument, and RNA quality was assessed using an Agilent TapeStation 2200 with
RNA ScreenTape (Agilent). Using 1 ug of total RNA input, stranded mRNA sample
preparation was performed with the Illumina TruSeq Stranded mRNA Library Prep
Kit (Illumina) following manufacturer’s protocol except the enzymatic
fragmentation time was reduced from 8 min to 1 min. Libraries were sequenced at
the Duke GCB Sequencing Core as 51 cycles paired-end runs (51PE), in an Illumina
HiSeq 4000 platform. Reads were aligned against the human reference genome
GRCh38 using the aligner STAR v2.4.1a45 following the proposed 2-pass strategy to first
identify a splice junction database to improve the overall mapping quality. Gene
counts were estimated with featureCounts from the subread package
v1.4.6-p646 , using
gene annotations from Refseq47 and allowing for multimapping reads (-M and --fraction arguments). Differential
expression analyses were performed using the DESeq2 package48 filtering out non-expressed genes and
fitting a negative binomial generalized linear model to find significantly
differentially expressed genes.