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Pipeline publication

[…] with 18 or more nucleotides remaining after the additional trimming were kept and processed as described below. The genes identified are in Additional file and were used in the comparisons in Fig. ., The fastq files were converted to fasta files, which were compressed to eliminate duplications, based on the tags. The compressed fasta file of tags was then separated into two files—representing the irrelevant and Mov10 immunoprecipitations—each file containing the tags with a specific barcode. This step utilized scripts that did the separation and also removed the barcode from the read, in preparation for the alignment step. The separated samples were aligned to the mouse genome (mm10) using Novoalign (RRID: SCR_014818). The only parameter specified was “-t 60”, this allows for two mismatches between the genome and the read. Uniquely mapping reads were extracted from the resulting sam files, and the information was converted to BAM format using samtools (samtools view; RRID: SCR_002105). The BAM format was converted to bed (genome interval) format using bedtools (version 2.17.0; RRID: SCR_006646) (bamToBed). The genome intervals of the reads (bed file) for each sample were merged into larger intervals using bedtools (mergeBed). The new interval/region is a location with a set of overlapping reads. Any regions that had any presence in the control Irrelevant samples (intersectBed) were removed to give the final set of experimental genome intervals that have reads mapping to them in the Mov10 immunoprecipitations, which will be referred to as “regions” from here on. IntersectBed (bedtools) was used to determine which regions overlap with genes, exons, UTRs, and lncRNAs (Ensembl) regions using bed files specific for these features respectively. All regions that overlap with genes, but not with exons, are considered intronic, and all other regions are considered intergenic. DAVID 6.8 analysis [, ] (RRID:SCR_001881) was performed on the Mov10 CLIP targets using a P1 C57Bl/6 brain background (Gene Expression Omnibus (GEO) numbers GSM417923, GSM417922, and GSM417921)., Total RNA from three P2 mice brains was used in the reverse transcription assay with either SuperScript III Reverse Transcriptase (Invitrogen ThermoScientific, Carlsbad, CA, USA) alone or with equimolar amounts of recombinant Mov10 or Fmrp [] after pre-incubating the proteins on ice for 5 min. cDNA synthesis was carried out at 50 °C for 45 min, then 70 °C for 15 min. RT-PCR reactions was carried out on the cDNA samples using primers to Prrc2b, mL1TF, or Gapdh (Additional file ). For qRT-PCR, iQ SYBR […]

Pipeline specifications

Software tools NovoAlign, SAMtools, BEDTools, DAVID
Databases GEO