|
| Status |
Public on Sep 26, 2022 |
| Title |
eiCLIP_sample2 |
| Sample type |
SRA |
| |
|
| Source name |
Primary Mouse Hepatocytes
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6/J
tissue: Liver
number of livers: 3
number of pellets: 3
rip antibody: Rbfox2 antibody (RBM9 - Bethyl Laboratories)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted as described and isolated with Rbfox2 antibody (RBM9 - Bethyl Laboratories). The protocol differed from the iCLIP protocol published previously (Sibley et al 2018. Individual Nucleotide Resolution UV Cross-Linking and Immunoprecipitation (iCLIP) to Determine Protein–RNA Interactions. In: Gaspar I. (eds) RNA Detection. Methods in Molecular Biology, vol 1649. Humana Press, New York, NY) by the following: The adaptor contains index 1 rather than the RT-primers thus allowing multiplexing of samples after adapter ligation. Excess adaptor is removed using recjf-deadnylase. RNA purification is carried out by column rather than overnigh ethanol precipitation. RT primer binds to the 3' of the adaptor and has a biotin attached enabling cDNA purification by Streptavidin beads. Free RT primer is ligated with a complementary primer and digested by exonuclease. There is no requirement for a ciruclarisation step. A solexa adaptor primer binds to the RT primer in advance of library preparation.
Library is constructed using Accuprime Supermix I and P5/P7 solexa primer mix. The P5/P7 primers are also indexed with independent barcodes. 22 cycles of PCR were used in this extraction method. Size selection of the cDNA occurs after library preparation using a zymogen select-a-size kit. The P7 primer has phosphthioate bonds between the last three nucleotides of the primer.
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
RNA bound to Rbfox2
|
| Data processing |
Illumina CASAVA 1.8.4 software used for basecalling.
Each raw read from the eiCLIP experiment contains a random barcode sequence in the first 7 nucleotides, where the 8th nucleotide is an A. The random barcode was moved to the header of the FASTQ file for the later removal of PCR duplicates and the 8th nucleotide was removed from the read sequence.
For the adapter removal of eiCLIP sequencing samples, we used ‘cutadapt’ tool (https://cutadapt.readthedocs.io/en/stable/) with the following parameters: ‘cutadapt -f fastq --match-read-wildcards --times 1 -e 0.1 -O 1 --quality-cutoff 6 -m 18 -a AGATCGGAAG $INPUT.fastq > $OUTPUT.adapterTrim.fastq 2> $OUTPUT.adapterTrim.metrics’.
eiCLIP samples were aligned to GENCODE assembly annotation version ‘GRCm38.VM20’ by STAR alignment tool (version 2.4.2a) (https://github.com/alexdobin/STAR) with the following parameters: ‘STAR –runThreadN 8 –runMode alignReads –genomeDir GRCm38 Gencode v20 –genomeLoad LoadAndKeep –readFilesIn read1, read2, –readFilesCommand zcat –outSAMunmapped Within –outFilterMultimapNmax 1 –outFilterMultimapScoreRange 1 –outSAMattributes All –outSAMtype BAM Unsorted –outFilterType BySJout –outFilterScoreMin 10 –alignEndsType EndToEnd –outFileNamePrefix outfile’.
For the overamplification correction of the eiCLIP samples we used a custom python script to swap random barcodes from the first 7 nts of the FASTQ sequence line to the FASTQ header line. Uniquely mapped reads with the same genomic positions and the same random barcode were then removed as PCR duplicates. At each cDNA-start position, the counts of all cDNAs were summed together and converted into in bedGraph format using a custom python script, where each number represents a unique number of crosslinking positions.
Then crosslink clusters were defined by using the cDNA-starts with counts as the input for False Discovery Rate clustering tool available by iMaps (https://imaps.genialis.com/iclip). The clusters were identified by using default parameters and Paraclu clustering algorithm (http://cbrc3.cbrc.jp/~martin/paraclu/).
Genome_build: mm10
Supplementary_files_format_and_content: bedGraph and BED6 format
|
| |
|
| Submission date |
Jun 03, 2020 |
| Last update date |
Sep 26, 2022 |
| Contact name |
LMS Bioinformatics Core |
| E-mail(s) |
bioinformatics@lms.mrc.ac.uk
|
| Organization name |
MRC London Institute of Medical Sciences
|
| Department |
Bioinformatics Core
|
| Street address |
Hammersmith Hospital Campus, Du Cane Road
|
| City |
London |
| ZIP/Postal code |
W12 0NN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL16417 |
| Series (2) |
| GSE151747 |
eiCLIP analysis of RBFOX2 in primary mouse hepatocytes |
| GSE151753 |
Characterization of the role of RBFOX2 in the liver |
|
| Relations |
| BioSample |
SAMN15096430 |
| SRA |
SRX8466165 |
