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Sample GSM4064593

Query DataSets for GSM4064593

Status

Public on Aug 26, 2022

Title

human WBC, GB4030128 (iDNase-seq)

Sample type

SRA

Source name

WBC

Organism

Homo sapiens

Characteristics

cell type: WBC

Extracted molecule

genomic DNA

Extraction protocol

DNase-seq human 1.25M 2-step crosslink human healthy T/B/NK/monocyte, final 0.1U/μl DNase digestion 37°C 5 minutes, T cell split #1-15 wells, B cell split #16-30 wells, NK cell split #31-45 wells, monocyte split #46-61 wells, TdT&T4 methods adding P7 barcode adaptor (12.5μl volumn/well 37°C 60 minutes), pool, dilution, counting and redistribute into 96-well plate, total 20 nuclei per well seal completely, 65°C overnight; direct PCR1 (12 cycles), pool all the DNA, EXO I 30 minutes, A-tailing and P5 ligation(rt overnight) , column purification and PCR2(16 cycles), run-e-gel and cut from 200-500bp rc#1 i5-01
The isolated 50M of PBMC suspended by 50ml PBS /MgCl2 are firstly fixed by adding 400μl freshly prepared 0.25M Disuccinimidyl glutarate (DSG, ThermoFisher Scientific, catalog no.20593) and incubate at room temperature for 45 minutes with rotation. After protein-protein fixation by DSG and washing three times by PBS, cells suspended by culture medium DMEM supplemented with 10% FBS are further fixed by the addition of 1:15 volume of 16% (w/v) methanol-free formaldehyde solution (Thermo Fisher Scientific) and incubate at room temperature for 10 minutes. The fixing reaction is terminated by a 1:10 volume of 1.25 M glycine and incubate at room temperature for 5 minutes. The fixed cells are collected by centrifugation at 1320rpm for 7 minutes and washed with PBS. The cells are stored in aliquots (1 × 10e6 cells per tube) at −80°C until use.

Library strategy

DNase-Hypersensitivity

Library source

genomic

Library selection

DNAse

Instrument model

Illumina HiSeq 3000

Data processing

Basecalls performed
The raw fastq files are read and separated into 480 files corresponding to 96 pairs of cell barcodes over 5 replicates. The 480 pairs are shown in barcode_480.txt. A read is considered as belonging to one of the barcodes if the beginning of read 1 or read 2 contains the same pair of barcodes in the left and right column respectively. After separting each set of fastq files into 480 files, some base pairs starting from 5' are trimmed and the remaining reads are mapped to the human genome using bowtie2.
The top 30 barcodes are selected from each well based on read count, so 30 nuclei are sorted for each well with doublets removed, where the number removed was calculated based on the collision rate like in (Science. 2015 May 22; 348(6237): 910–914). These are denoted as single cells because of the low likelihood of two cells in the same well having the same barcode. We required that qualifying cells have more than 100 reads and a FRiP greater than 0.1. With these requirements, 4489 cells were obtained, and we were able to identify 218,042 DHSs. For each cell, we calculated the reads that were located within the DHS and stored the results in a matrix.
Genome_build: hg18
barcode_480.txt: two columns, the left column refers to a DNA barcode starting from the 5' end while the right column refers to a DNA barcode starting from the 3' end
peaks_218042_WBC.txt: first column is for chromsome, second column is for DHS start site, and third column is for DHS end site
iDNase_4489_cells_218042_peaks_counts_at_WBC.mtx: This file contains read count data for single cells in MatrixMarket format. Each row corresponds to a non-zero entry with the row, column, and then value specified. Reading the matrix results in a 218042 x 4489 matrix of peaks by cells where each element represents the number of reads located within a DHS site for some single cell.

Submission date

Sep 06, 2019

Last update date

Aug 26, 2022

Contact name

Keji Zhao

E-mail(s)

[email protected]

Phone

301-496-2098

Organization name

NHLBI