/Efficient-RNA-polymerase-II-pause-release-requires-U2-snRNP-function_2021

Caizzi, Monteiro-Martins (2021)

Primary LanguageR

Efficient RNA-polymerase II pause release requires U2 snRNP function (2021)

Fastq files and annotation used on this analysis are available from GEO series GSE148433.

Pre-analysis:

TT-seq and RNA-seq reads were aligned using STAR and filtered with Samtools. HTSeq was used to calculate the read counts for different featues (please see https://github.com/cramerlab/TT-seq_analysis)

Annotation:

We included in our analysis only major isoforms with 70% or higher prevalenve per gene in both DMSO and Pla-B. In order to create the major isoforms annotation:

Create general annotation:

Download ncbi Refseq hg38 genome assembly from https://genome.ucsc.edu/cgi-bin/hgTables
(clade: Mammal, genome: Human, assembly: Dex.2013 GRCh38/h38, group: Gene and Gene Predictions, track: NCBI RefSeq, table: RefSeq Curated)
R_scripts/create_anno.R
R_scripts/create_extended.R

Create major isoform annotation:

bash/salmon.sh
R_scripts/major_isoform_selection.R.

Create intronless genes annotation:

R_scripts/intronless_annotation.R

Splicing ratio calculation:

  1. Create Exon based spliced junction read count lists using bam files:
    R_scripts/exon_based_spliced_junction.R
  2. Calculate the splicing ratio and plot the results:
    R_scripts/splicing_ratio.R

Splicing-affected and unaffected transcripts identification:

R_scripts/splicing_affected_unaffected.R

Elongation velocity calculation:

  1. Create Unique Transcribed Bases Rle Tracks for TT-seq and Fragment End Rle Tracks for mNET-seq: R_scripts/TTseq_coverage_rle_tracks.R
    R_scripts/mNETseq_coverage_rle_tracks.R
  2. Create coverage files from rle tracks:
    (for mNET seq create coverage using ProcessedData/mNETseq/FragmentEndRleTracks)
    R_scripts/coverage.R
  3. Calculate the RNA amount pre cell (based on TT-seq spike-ins coverage):
    R_scripts/RNA_amount_per_cell.R
  4. Calculate the elongation velocity using TT-seq and mNET-seq normalized coverage:
    R_scripts/elong_velocity.R