5 protocols
normalization data transformation protocol
For PAR-CLIP, all PAR-CLIP sequencing data were analyzed with a computational analysis pipeline (Lebedeva, S. et al. 2011 Mol Cell 43, 340-52).Illumina PAR-CLIP cDNA sequencing reads were aligned to the mouse genome assembly (mm9) allowing for up to one mismatch, insertion or deletion. Only uniquely mapped reads were retained. We identified the clusters of aligned reads continuously covering genomic regions based on the condition that a sequence cluster requires at least one T-to-C conversion. The number of T-to-C mismatches served as a crosslink score, and the position with the highest frequency of T-to-C conversion was referred to as the preferred crosslinking position. We also assigned a quality score based on the number and positions of distinct reads contributing to the cluster. As the reads should originate from protein-bound transcripts we regarded clusters aligning antisense to the annotated direction of transcription as false positives. We were thus able to select cutoffs on both scores such as to keep the estimated false positive rate below 5%. After filtering by these cutoffs we expect each remaining cluster to harbor at least one binding site. For RNA-seq, all obtained reads were mapped to the mm9 genome sequence with TopHat (v1.1.2) with options -p 6 -a 5 -g 1 --segment-length 30, then use Cufflink (v1.3.0) to estimate the isoform FPKM-levels with options -p 6 using the aligned reads and the RefSeq gene models. Theprocessed data (_cufflink.txt) files contain FPKM values. Genome_build: mm9 Supplementary_files_format_and_content: bed and txt
nucleic acid library construction protocol
For RNA-seq, Polyadenylated RNA from the mock cells at 37°C or 32°C with two replicates, siCirbp-1, siCirbp-2, siRbm3-1 and siRbm3-2 MEFs at 37°C were sequenced on Illumina GAII using 76 bp single-end kits according to the manufacturers instructions.
sample treatment protocol
For PAR-CLIP, the MEFs were incubated with 4-thiouridine (4SU) for 16 h before crosslinking. For RNA-seq, no treatment.
growth protocol
The MEFs were cultured in DMEM medium with 10% FBS at 37°C
nucleic acid library construction protocol
For PARCLIP, the experiment was performed as in Hafner et. al 2010 Cell, but with an antibody against Flag (Sigma) in MEFs. The MEFs that are stably expressing the FLAG epitope-tagged protein of interest were grown in medium supplemented with 100µM 4SU for 16h prior to harvest.