GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3587036

Query DataSets for GSM3587036

Status

Public on Aug 20, 2019

Title

human HEK293T H3K4me3 - 23

Sample type

SRA

Source name

HEK293T

Organism

Homo sapiens

Characteristics

cell lines: HEK293T

Extracted molecule

genomic DNA

Extraction protocol

HEK293T cells were washed with PBS and permeabilized using a 10 min incubation on ice in 500 mL of complex formation buffer per ~1 million cells. After permeabilization, all subsequent centrifugations of cells were performed using the following procedure: 1 min spin at 250 *g, rotate tubes 180°, repeat spin, leave ~10 mL of solution in the tube when removing buffer. Pelleted cells were washed once with 500 mL of complex formation buffer and suspended in another 500 mL of buffer. Aliquots of 1,000 cell equivalents were transferred volumetrically to clean microcentrifuge tubes and adjusted to 50 mL total volume using complex formation buffer. 5 mL of antibody-bound PA-Tnp complex was added to each 50 mL cell aliquot and incubated at room temperature for 60 min to allow chromatin binding. Unbound complex was removed using three washes performed as follows: pellet and suspend cells in 500 mL of wash buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% Triton X-100), rotate tube for 8 min at room temperature, repeat. Pelleted cells were rinsed with 500 mL of rinse buffer (50 mM Tris pH 8.0, 50 mM NaCl, 0.1% Triton X-100). The rinse buffer was removed and 90 mL of reaction buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10 mM MgCl2, 0.1% Triton X-100) was added to the pelleted cells for a total volume of ~100 mL. The tubes were incubated for 30 min at 37 °C to allow transposition to occur. The reaction was terminated by addition of 4 mL of 0.5 M EDTA, 2 mL of 10% SDS, and 1 mL of 20 mg/mL Proteinase K followed by a 60 min incubation at 65 °C.
DNA was purified using phenol-chloroform-isopentanol extraction and ethanol precipitation. The DNA pellet was suspended in 10 mL of 10 mM Tris pH 8.0. PCR reactions for library preparation were performed by adding the following to the 10 mL DNA sample: 25 mL of Phusion High-fidelity PCR Master Mix (NEB catalog # M0531S), 1 mL of 5¢ library index barcode, 1 mL of 3¢ library index barcode, and 13 mL of nuclease-free water. Amplification was performed using an initial step of 72 °C for 5 min followed by 15 cycles of: 98 °C for 10 s, 65 °C for 30 s, 72 °C for 15 s, and a final extension step of 72 °C for 5 min. The PCR products were analyzed using agarose gel electrophoresis. Fragments of the desired size were excised and purified using a QIAquick Gel Extraction kit (Qiagen cat # 28506).
scACT-seq

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 3000

Description

scACT-seq

Data processing

Basecalls performed using RTA 1.7 or Illumina Casava1.7
Read in the raw fastq files are separated into 96 files correponding to the 96 pairs of cell barcode. The 96 pairs of barcodes are shown in the file named barcode_96.txt. A read is considered to belong to one of the barcode if the beginning of correpsonding read1 or read 2 contain the same pair of barcodes in theleft and right column respectively. After separting each set of fastq files into 96 set of files, the 34bp starting from 5' is trimmed and the remainging reads are mapped to human genome using bowtie2.
Since 18 nuclei is sorted for each well, we removed doublets and the top 14 ranked barcodes are considered to be cells. The doublets and the number of 14 were calculated based on the collision rate the same as in the reference (Science. 2015 May 22; 348(6237): 910–914. ) . With requiring the qualified cells have reads more than 500, 1246 single cells are obtained. On the other hand, Using the publsihed HEK293T DNase-seq data, we identified 15756 DHS (as shown in the file called peaks_15756_HEK293T.txt). For each cell, we calculated the reads that were located within the DHS. Finally, we obtained a read count file for the single cells, in which the 1246 columns referred to single cells, and the 15756 rows refer to 15756 DHS regions.
Genome_build: hg18
Supplementary_files_format_and_content: barcode_96.txt: 2column. The left column refer to barcode on read 1 starting from the 5' end while The right column refer to barcode on read 1 starting from the 5' end
Supplementary_files_format_and_content: peaks_15756_HEK293T.txt: the first column is chromsome, 2nd column is the start of DHS, 3rd column is the end of DHS
Supplementary_files_format_and_content: scH3K4me3_1246_cells_counts_at_293T.txt: This file contain the read count data of single cells. Each column refer a single cell and each row refer to a DHS, resulting in a matrix of 15756 x 1246. Each element in the matrix represent the number of reads from the single cell located within the DHS.

Submission date

Jan 31, 2019

Last update date

Aug 20, 2019

Contact name

Keji Zhao

E-mail(s)

zhaok@nhlbi.nih.gov

Organization name

national Institute of Health

Department

National Heart, Lung, and Blood Institute