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| Status |
Public on Jun 05, 2023 |
| Title |
flag-hct116-orc1-oe-highrnase-up-rep2 |
| Sample type |
SRA |
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| Source name |
Colorectal Cancer
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
treatment: Transfected with pcDNA3.1-ORC1-3xFLAG
antibody: FLAG antibody (M2, F1804, Sigma)
replicate: 2
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| Treatment protocol |
48 hours transfection of pcDNA3.1-ORC1-3xFLAG.
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| Growth protocol |
HCT116 cells were grown in RPMI medium at 37ºC.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The iCLIP protocol was followed to isolate RNA bound to ORC1-3xFlag and amplify a cDNA library as described in Mas et al. 2021.
Adapter ligation was performed after immunoprecipitation and samples for each condition were barcoded during RT, using RT primers with specific barcodes. Random nucleotides (UMIs) were also incorporated into cDNAs to distinguish between PCR duplication of cDNA products.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
On the iCLIP gel isolation cut upper band than expected ORC1 molecular weight, with High RNase treatment Replicate 2
Flag_HCT116_ORC1-OE_HighRNase_Up_merge_grouped_cdna.bed
Flag_HCT116_ORC1-OE_HighRNase_Up_merge_grouped_cdna_peaks.bed
Flag_HCT116_ORC1-OE_HighRNase_Up_merge_grouped_cdna_norm.bigWig
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| Data processing |
Library strategy: iCLIP-seq
iCLIP sequencing reads were analysed on the iMaps server (https://imaps.genias.com/) using the iCount software (https://github.com/tomazc/iCount 18).
Briefly, experimental barcodes were removed and sequencing reads aligned with STAR 9 to human genome build GRCh38 primary assembly GENCODE version 27, allowing two mismatches and ten secondary alignments.
Unique Molecular Identifiers (UMIs), were used to distinguish and remove PCR duplicates. To determine protein-RNA contact sites, the sequencing read preceding nucleotide was allocated as the crosslink site event.
Significant contact sites were then identified, using the iCount peak function, based on false discovery rate (FDR) <0.05 comparing specific sites within a window of three nucleotides with randomised data (100 permutations) and within co-transcribed regions (https://github.com/tomazc/iCount/blob/master/iCount/analysis/peaks.py). The significant crosslink signal was normalised by CPM. For individual samples we also used Paraclu peak caller.
Assignment of crosslink sites to coding transcripts, non-coding or biotype features, was done by following segmentation hierarchy rules (https://github.com/tomazc/iCount/blob/master/iCount/genomes/segment.py).
Then, replicates were merged and summary of cDNA counts within genes and genic regions were generated with iCount summary function, normalising the counts by the length of the corresponding region.
Genome_build: GRCh38 primary assembly GENCODE version 27
Supplementary_files_format_and_content: Crosslink bed, group bed, peaks bed, group peaks bed, normalised bigwig.
Supplementary_files_format_and_content: flag-hct116-notf-down_trimmed_single.bed: crosslink bed
Supplementary_files_format_and_content: flag-hct116-notf-up_trimmed_single.bed: crosslink bed
Supplementary_files_format_and_content: flag-hct116-orc1-oe-down-rep2_trimmed_single.bed: crosslink bed
Supplementary_files_format_and_content: flag-hct116-orc1-oe-down-rep3_trimmed_single.bed: crosslink bed
Supplementary_files_format_and_content: flag-hct116-orc1-oe-highrnase-up-rep1_trimmed_single.bed: crosslink bed
Supplementary_files_format_and_content: flag-hct116-orc1-oe-highrnase-up-rep2_trimmed_single.bed: crosslink bed
Supplementary_files_format_and_content: flag-hct116-orc1-oe-up-rep1_trimmed_single.bed: crosslink bed
Supplementary_files_format_and_content: flag-hct116-orc1-oe-up-rep2_trimmed_single.bed: crosslink bed
Supplementary_files_format_and_content: flag-hct116-orc1-oe-up-rep3_trimmed_single.bed: crosslink bed
Supplementary_files_format_and_content: flag-hct116-notf-down_trimmed_single_peaks.bed: peaks bed
Supplementary_files_format_and_content: flag-hct116-notf-up_trimmed_single_peaks.bed: peaks bed
Supplementary_files_format_and_content: flag-hct116-orc1-oe-down-rep2_trimmed_single_peaks.bed: peaks bed
Supplementary_files_format_and_content: flag-hct116-orc1-oe-down-rep3_trimmed_single_peaks.bed: peaks bed
Supplementary_files_format_and_content: flag-hct116-orc1-oe-highrnase-up-rep1_trimmed_single_peaks.bed: peaks bed
Supplementary_files_format_and_content: flag-hct116-orc1-oe-highrnase-up-rep2_trimmed_single_peaks.bed: peaks bed
Supplementary_files_format_and_content: flag-hct116-orc1-oe-up-rep1_trimmed_single_peaks.bed: peaks bed
Supplementary_files_format_and_content: flag-hct116-orc1-oe-up-rep2_trimmed_single_peaks.bed: peaks bed
Supplementary_files_format_and_content: flag-hct116-orc1-oe-up-rep3_trimmed_single_peaks.bed: peaks bed
Supplementary_files_format_and_content: Flag_HCT116_NoTF_Up_Down_merge_grouped_cdna.bed: group bed
Supplementary_files_format_and_content: Flag_HCT116_ORC1_down_merge_grouped_cdna.bed: group bed
Supplementary_files_format_and_content: Flag_HCT116_ORC1-OE_HighRNase_Up_merge_grouped_cdna.bed: group bed
Supplementary_files_format_and_content: Flag_HCT116_ORC1_up_merge_grouped_cdna.bed: group bed
Supplementary_files_format_and_content: Flag_HCT116_NoTF_Up_Down_merge_grouped_cdna_peaks.bed: group peaks bed
Supplementary_files_format_and_content: Flag_HCT116_ORC1_down_merge_grouped_cdna_peaks.bed: group peaks bed
Supplementary_files_format_and_content: Flag_HCT116_ORC1-OE_HighRNase_Up_merge_grouped_cdna_peaks.bed: group peaks bed
Supplementary_files_format_and_content: Flag_HCT116_ORC1_up_merge_grouped_cdna_peaks.bed: group peaks bed
Supplementary_files_format_and_content: Flag_HCT116_NoTF_Up_Down_merge_grouped_cdna_norm.bigWig: normalised bigwig
Supplementary_files_format_and_content: Flag_HCT116_ORC1_down_merge_grouped_cdna_norm.bigWig: normalised bigwig
Supplementary_files_format_and_content: Flag_HCT116_ORC1-OE_HighRNase_Up_merge_grouped_cdna_norm.bigWig: normalised bigwig
Supplementary_files_format_and_content: Flag_HCT116_ORC1_up_merge_grouped_cdna_norm.bigWig: normalised bigwig
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| Submission date |
Apr 27, 2021 |
| Last update date |
Jun 05, 2023 |
| Contact name |
Maite Huarte |
| E-mail(s) |
maitehuarte@unav.es
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| Organization name |
CIMA
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| Street address |
c/ Avd Pio XII, 55
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| City |
Pamplona |
| State/province |
Navarra |
| ZIP/Postal code |
31008 |
| Country |
Spain |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE173449 |
ORC1 binds to cis-transcribed RNAs for efficient activation of human origins [ORC1_iCLIP] |
| GSE173452 |
ORC1 binds to cis-transcribed RNAs for efficient activation of human origins |
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| Relations |
| BioSample |
SAMN18883281 |
| SRA |
SRX10691379 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5268063_flag-hct116-orc1-oe-highrnase-up-rep2_trimmed_single.bed.gz |
6.5 Mb |
(ftp)(http) |
BED |
| GSM5268063_flag-hct116-orc1-oe-highrnase-up-rep2_trimmed_single_peaks.bed.gz |
15.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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