GSE155649

Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [RNA-seq]

1. Raw reads were trimmed using cutadapt (v1.14) using the following parameters -O 5 -f fastq --match-read-wildcards --times 2 -e 0.0 --quality-cutoff 6 -m 18 -o data.fastqTr.fq -b TCGTATGCCGTCTTCTGCTTG -b ATCTCGTATGCCGTCTTCTGCTTG -b CGACAGGTTCAGAGTTCTACAGTCCGACGATC -b GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -b AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA -b TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT data.fastq.gz
2. Trimmed reads were mapped to and filtered of repeat elements (RepBase 18.05) with STAR (2.4.0i) using the following parameters: --alignEndsType EndToEnd --genomeDir repbase --genomeLoad NoSharedMemory --outBAMcompression 10 --outFileNamePrefix data --outFilterMultimapNmax 10 --outFilterMultimapScoreRange 1 --outFilterScoreMin 10 --outFilterType BySJout --outReadsUnmapped Fastx --outSAMattrRGline ID:foo --outSAMattributes All --outSAMmode Full --outSAMtype BAM Unsorted --outSAMunmapped Within --outStd Log --readFilesIn data.fastqTr.fq --runMode alignReads --runThreadN 8
3. Reads unmapped to repeat elements were mapped to the human genome with STAR using the same parameters as the previous step, using an hg19 index in place of the repeat element index
4. Subread featureCounts (-a gencode.v19.annotation.gtf -s 2 -p -o counts.txt data.bam) was used to count features using human annotations (Gencode v19)
5. Edits were called using SAILOR (http://github.com/yeolab/sailor) using default parameters and dbSNP (v147) to remove known SNPs.
6. bigwig files were generated from filtered BAM files (intermediates from SAILOR), using the following commands from BedTools (v2.27.1). : bedtools genomecov -split -strand - -g hg19.chrom.sizes -bg -ibam fwd.bam > fwd.bg bedtools sort -I fwd.bg > fwd.sorted.bg bedGraphToBigWig fwd.sorted.bg hg19.chrom.sizes fwd.sorted.bw bedtools genomecov -split -strand + -g hg19.chrom.sizes -bg -ibam rev.bam > rev.bg bedtools sort -I rev.bg > rev.sorted.bg bedGraphToBigWig rev.sorted.bg hg19.chrom.sizes rev.sorted.bw

Supplementary Files (10)

Dataset Citations (2)

Linked Publications (3)