title: GSM4727011: RNA-seq_high_MYC_replicate2; Mus musculus; RNA-Seq
library_construction_protocol: RNA-seq: After sorting, the low-MYC and high-MYC cells were seeded overnight. Considering the effects of MYC on global gene expression, external RNA spike-in control was added to cells in proportion to cell count before total RNA extraction. The mRNA libraries were prepared using TruSeq Stranded mRNA (Illumina) and sequenced on a NextSeq sequencer. ChIP-seq: Primary MEFs were cross-linked with 1% formaldehyde for 8 min (FUBP1 ChIP) and the cross-linking was terminated by adding glycine to a final concentration of 0.125M. After harvesting cells, the cell pellets were resuspended in RIPA buffer supplemented with protease inhibitors. Samples were sonicated to produce chromatin fragments of 300 bp on average, and subjected to immunoprecipitation. Five μg of rabbit monoclonal anti-FUBP1 antibody were used for the immunoprecipitation. The immunoprecipitated samples were subjected to library preparation and sequencing. TACh-seq: MEFs were lysed in hypertonic lysis buffer and digested with Cyanase (RiboSolutions) for 3 min at 37 degree. Reactions were terminated by the addition of EDTA (10 mM final) and SDS (0.75% final). After RNA and protein digestion, DNA was purified and the fragments of 100- 600 bp were selected by gel electrophoresis. Libraries were prepared using the manufacturer's protocols. RNA-seq:After trimming bases on the 3' end with quality scores below 20, reads were aligned to the mouse transcriptome (mm10) using TopHat. For each ERCC RNA, the number of reads mapping to that transcript was divided by the transcript length to yield RPK (reads per kilobase of transcript). From the top 50 expressed transcripts as measured by average RPK over all samples, a set of 10 highly correlated transcripts was used to represent the quantity of spike-in RNA in each sample. The normalization score for each sample was then calculated as the geometric mean of the expression (FPK) of those 10 genes in that sample. Reads mapped to the transcriptome were quantified using the Partek E/M algorithm. For each transcript, the number of reads quantified to that transcript was divided by the transcript length to yield RPK, then divided by the normalization score for that sample to normalized to the number of cells. The normalized FPK of low-MYC and high-MYC cells were compared. ChIP-seq, TACh-seq