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Sample GSM2322540

Query DataSets for GSM2322540

Status

Public on Nov 15, 2016

Title

STL001_Bladder Rep1

Sample type

SRA

Source name

Bladder Tissue

Organism

Homo sapiens

Characteristics

tissue: Bladder Tissue

Extracted molecule

genomic DNA

Extraction protocol

For newly generated Hi-C datasets presented in this study, Hi-C was performed as previously described (Lieberman-Aiden et al., 2009) with minor modifications pertaining to the handling of flash-frozen human tissue as previously described (Leung et al., 2015). Flash frozen human tissues were first pulverized using mortar as pestle, and then crosslinked for 25min using 1% formaldehyde, quenched with Glycine, washed with PBS and then input into the remaining Hi-C protocol or flash frozen for future analysis. For each experiment, ~65mg of tissue was used for subsequent Hi-C library preparation, but varied from tissue to tissue depending on cellular density of each tissue. Using ~65mg of tissue, samples were lysed and dounced to obtain single nuclei suspensions. Chromatin was then solubilized with SDS, and then quenched with TritonX-100. Chromatin was digested overnight with HindIII. Digested ends were filled-in dNTPs, except a biotinylated dCTP was used to label digested ends. Nuclei were then lysed using SDS, and chromatin complexes were diluted. Ligation was performed in large volumes to favor intra-molecular ligation events. Crosslinks were reversed by overnight incubation with Proteinase K and high temperatures. DNA was then purified using two rounds of Pheno-Chloroform purification, followed by ethanol precipitation. Biotinylated nucleotides on unligated ends were removed by a T4 DNA polyermase reaction, and purified using ethanol precipitation. DNA was then quantified using Qubit Fluorometer, and <=5ug of DNA was used for subsequent library preparation.
Libraries were constructed using a modifed Illumina TruSeq library preparation method specific to the preparation of Hi-C libraries for Illumina sequencing, as previously described (Lieberman-Aiden et al, 2009). First Hi-C ligation products were sheared to ~400bp fragment size. Sheard ends were repaired using a combination of T4 PNK, T4 DNA polymerase, and Klenow. The reaction was purified using Qiagen MinElute columns. DNA molecules were then dA-tailed using Klenow(exo-) and then size selected from 200-600bp using agaorse gel and purified. Biotin-labeled DNA species were then enriched using streptavidin-coated beads and Illumina TruSeq adapters were ligated using T4 DNA ligase while DNA is bound to the streptavidin beads. Beads were then washed, and PCR amplified to obtain enough material for Illumina sequencing.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

STL001_Bladder Rep1

Data processing

Library strategy: HiC-Seq
Each Hi-C read end was mapped indepenedently using BWA -mem and default parameters. BWA version 0.7.8 was used.
Read ends with >1 alighment were filtered and only 5' alignments were kept using in-house Python script.
SAM files were converted to BAM files and sorted using Samtools Version 0.1.18
PCR duplicates were removed using PicardTools Version 1.49
Reads aligning >500bp from Hi-C restriction enzyme cutsites were removed and reads <15kb insert size were removed using in-house scripts.
Raw contact matrices were constructed using in-house scripts, or normalization was applied using HiCNorm, VC, or ICE, where noted.
FIREs were identified by first applying HiCNormCis on the raw contact marix, as described in this manuscript.
TAD boundaires were identified by applying the insulation square method on the HiCNorm matrices, as previously described (Crane et al., 2015)
A/B compartments were identified as previously described (Lieberman-Aiden et al., 2009)
Statistically significant Hi-C contacts were identified using Fit-Hi-C, as previously described (Ay et al., 2014)
Genome_build: Human data is in hg19, mouse data is in mm9.
Supplementary_files_format_and_content: Hi-C matrices are provided at 40kb bin resolution in NxN format for each chromosome for either raw contact maps, or Hi-C matrices normalized with HiCNorm, or HiCNorm followed by quantile normalization using samples from the same published or unpublished source. Even if a dataset was not specifically used in this study, a contact matrix was generated for each sample, for each study. For example, all samples from Zuin et al, 2014 are quantile normalized together. FIRE and TAD boundary annotations are provided as a unique BED file for each sample. Compartment A/B annotations are provided as a unique bigwig file for each sample. Fit-Hi-C output is provided in a 7-column format, corresponding to position 1, position 2, observed count, expected count, O/E, p-value, and q-value. Only the upper-triangle matrix entries (where i<=j) are provided, and only pairwise contacts within 2Mb genomic distance are provided.

Submission date

Sep 20, 2016

Last update date

Feb 22, 2021

Contact name

Anthony D Schmitt

E-mail(s)

[email protected]

Phone

6178429022

Organization name

UC San Diego