This repository includes scripts accompanying the study on RNA porcessing in the fission yeast S. pombe by long-read sequencing
Long-read SMRT cDNA sequencing of nascent RNA from exponentially growing S. pombe 972h- and prp2-1 cells first at 26C and then for 2 hours at 37C was employed to obtain splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010). The nascent 3’ end was labeled with a 3’ DNA adaptor through ligation. The adaptor sequence served as template for full-length reverse transcription and double-stranded cDNA was obtained in a transcriptome-wide PCR. The dataset contains data from 3 replicates per strain. Scripts for demultiplexing the samples, filtering for unique barcode matching and removal of transcripts with short polyA tails are deposited here.
Sample barcodes (for dataset published http://dx.doi.org/10.7910/DVN/PW1KEG) are: sample_id barcode barcode_reverse_complement; SP_wt_BC1 TCAGACGATGCGTCAT ATGACGCATCGTCTGA; SP_wt_BC2 CTATACATGACTCTGC GCAGAGTCATGTATAG; SP_wt_BC3 TACTAGAGTAGCACTC GAGTGCTACTCTAGTA; SP_prp2_BC4 TGTGTATCAGTACATG CATGTACTGATACACA; SP_prp2_BC5 ACACGCATGACACACT AGTGTGTCATGCGTGT; SP_prp2_BC6 GATCTCTACTATATGC GCATATAGTAGAGATC; spike_in_BC7 ACAGTCTATACTGCTG CAGCAGTATAGACTGT;
The polyA tail filtering process requires an input bed file, which provides the polyA site annotation of genes plus 100 nt up- and downstream of the annotated site.