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Sample GSM1281297

Query DataSets for GSM1281297

Status

Public on Apr 30, 2023

Title

CHH2430

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

Technical 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

E-mail(s)

Oscar.Luo@jax.org

Organization name

The Jackson Laboratory for Genomic Medicine

Street address

10 Discovery Dr