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Sample GSM1281299 Query DataSets for GSM1281299
Status Public on Apr 30, 2023
Title JCHH2431
Sample type SRA
Source name HeLa S3
Organism Homo sapiens
Characteristics cell line: HeLa S3
Extracted molecule genomic DNA
Extraction protocol Approximately 10^8 HeLa S3 cells were subjected to dual-crosslinking with 1% formaldehyde and 1.5 mM EGS. Cells were harvested and lysed. Complexes of lncRNA-chromatin with protein were sheared to fragments of approximately 500 bp, which then were biotinylated with EZlink Iodoacetyl-PEG2-Biotin (IPB). The streptavidin beads-bound lncRNA-chromatin-protein complexes were subjected to RICh-PET library construction. The DNA fragments present in streptavidin beads-bound chromatin were end-repaired using T4 DNA polymerase. The chromatin complexes were split into two aliquots, followed by the addition of RNA linkers with different barcode sets modified with biotin and containing flanking MmeI restriction sites, RNA linker “R1” (tube1) and RNA linker “R2” (tube2). Linkers to the RNA end were annealed, first-strand cDNA was synthesized, and relative DNA linkers “D1” (tube 1) and DNA linker “D2” (tube 2) were ligated. The two tubes were mixed together for proximity ligation. Chromatin-associated RNA fragments with linkers were subjected to second-strand cDNA synthesis. The cDNA-DNA PETs were extracted from the ligation products by MmeI digestion. Released biotinylated PETs were purified by streptavidin-coated magnetic beads, ligated to adaptors, and scaled up by PCR. Gel-purified amplicons of PET templates were sequenced using an Illumina® HiSeq 2500 with paired-end sequencing.
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2500
Description Biological replicate
Data processing Library strategy: RICh-PET
Basecalls and demultiplexing performed using CASAVA version 1.8.
PET reads from all three sequenced libraries were combined into one dataset.
PET reads were first filtered and trimmed for linker barcodes to identify the DNA sequence tag and RNA sequence tag, then mapped to the reference genome by BatMis. Only uniquely mapped reads on both ends were retained.
Redundant reads were removed. Then PET reads mapping location were extended 500bp upstream on both the DNA and RNA tags for downstream analysis. See the Supplemental information of the paper for detailed data processing procedures.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited file documenting the DNA and RNA tag genomic mapping location after 500bp extension.
Submission date Dec 06, 2013
Last update date Apr 30, 2023
Contact name Oscar J. Luo
Organization name The Jackson Laboratory for Genomic Medicine
Street address 10 Discovery Dr
City Farmington
State/province CT
ZIP/Postal code 06030
Country USA
Platform ID GPL16791
Series (1)
GSE53052 Comprehensive RNA-Chromatin Interactome Reveals Global and Specific Regulatory Functions by Long Non-Coding RNAs
BioSample SAMN02437464
SRA SRX387846

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

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