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Sample GSM3154189

Query DataSets for GSM3154189

Status

Public on Jun 07, 2018

Title

mES-DNA-SPRITE03

Sample type

SRA

Source name

Mouse Embryonic Stem cells

Organism

Mus musculus

Characteristics

cell line: F1 2-1
protocol: DNA SPRITE

Growth protocol

Mouse ES cell lines were cultured in serum-free 2i/LIF medium and maintained at an exponential growth phase

Extracted molecule

genomic DNA

Extraction protocol

Cells were crosslinked with 3%FA-DSG and then lysed with nuclei isolated. DNA was then lightly sonicated and DNAse digested to an average size of approximately 150-1000 bp in length. Crosslinked protein-DNA complexes were isolated on NHS beads, DNA ends were repaired, dA-tailed to generate a protruding 3’ A-base for ligation of barcodes, and were then split-pool tagged with a series of barcodes over multiple rounds of split-and-pool tagging. For DNA-RNA SPRITE, a distinct RNA adaptor was ligated to RNA to flag a molecule as RNA rather than DNA. This adaptor contains a shared overhang that allows for simultaneous tagging of RNA and DNA during the split-and-pool tagging process. Reverse transcription was performed to convert RNA into cDNA. After split-and-pool tagging, crosslinks were reversed, the DNA/RNA was isolated, was amplified 8-12 cycles using standard Illumina library prep (single index), and purified using SPRI beads. Libraries were sequenced on the HiSeq2500 or NextSeq 500 following the manufacturer's protocols.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

The reads have the following structure: Read 1 contains genomic DNA positional information and the DPM tag, and Read 2 has the remaining tags.
Processed data files:
mouse.mapq-ge10.clusters.gz
mouse.mapq-ge30.clusters.gz

Data processing

Library strategy: DNA SPRITE
SPRITE barcodes were identified by parsing the first DNA tag sequence from the beginning of Read 1 (DPM) and the remainder of the tags were parsed from Read 2. We identified these tag sequences corresponding to the DPM, Odd, Even, and Terminal tags that were added to these samples. The tags and their sequences were identified by software available at: https://github.com/GuttmanLab/sprite-pipeline/wiki/1.-Barcode-Identification. The configuration files required for this software are available on the series record.
We aligned each read to the appropriate reference genome (mm9 for mouse; hg19 for human) using Bowtie2 (v2.3.1). We trimmed the 11 base pair tag sequence (DPM) from Read 1 using the --trim5 11 parameter and used a local alignment search (--local).
We filtered the alignments for low quality reads, multimappers, and repetitive sequences. First, we removed all alignments with a MAPQ score less than 10 or 30. Second, we removed all reads that had >2 mismatches to the reference genome. Third, we removed all alignments that overlapped a region that was masked by Repeatmasker (UCSC, milliDiv < 140) using bedtools (v2.26.0). Fourth, we removed any read that aligned to a non-unique region of the genome by excluding alignments mapping to regions generated by the ComputeGenomeMask program in the GATK package (readLength=35nt mask).
To define SPRITE clusters, all reads that have the same barcode sequence were grouped into a single cluster. To remove possible PCR duplicates, all reads starting at the same genomic position with identical barcodes were removed.
Genome_build: mm9 (mouse), hg19 (human)
Supplementary_files_format_and_content: Cluster files were generated for the F1-21 mES cell and GM12878 lymphoblast DNA-SPRITE samples for all reads with MAPQ scores greater than 10 or 30. Each cluster, containing all reads with a shared series of tags, occupies one line of the resulting text file and contains the barcode name and genomic alignments. When multiple positions are listed, these regions are in physical contact with each other. If only one position is listed, either that fragment exists on its own as a singleton, or the other constituent pieces were not sequenced.

Submission date

May 24, 2018

Last update date

Jun 07, 2018

Contact name

Sofia Agustina Quinodoz

Organization name

Princeton University

Department

CBE

Lab

Brangwynne Laboratory