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Sample GSM2395418

Query DataSets for GSM2395418

Status

Public on Feb 01, 2021

Title

HeLa Nucleolus Sequencing rep2

Sample type

SRA

Source name

HeLa Nucleolus Sequencing biological repeat 2

Organism

Homo sapiens

Characteristics

cell line: HeLa
cell type: cervical cancer

Treatment protocol

Cells were treated with ActD (10 ug /ml) for 8 hours and then harvested to perform Hi-C and RNA-seq experiments.

Growth protocol

HeLa and U2OS cells were cultured in DMEM medium (ATCC-30-2001) with 10% fetal bovine serum and 5% CO2 at 37°C.

Extracted molecule

genomic DNA

Extraction protocol

Nucleoli isolation. Resuspend 2 – 5 x 106 cells with 1.5 ml 0.5% sodium dodecyl sulfate (SDS) and incubate at 62℃ for 5-10 min in order to dissolve the cell membrane. Then add 1.5 ml buffer S1 (0.25 M sucrose and 10 mM MgC12), mix well, and break the cells with ultra-sonication. Monitor the cells under a microscope and stop when over 80% of the cells are burst, leaving intact nucleolus with cytoplasmic materials. Transfer the liquid to a 15 ml tube, carefully add 3 ml buffer S2 (0.35M sucrose, 0.5mM MgC12) to the bottom of the tube and keep the two layers cleanly separated. Centrifuge at 3000 rpm for 5 min at 4℃ and discard the supernatant. Resuspend the pellet with 3 ml buffer S2, filtered with 5 um filter (Millipore) and transferred to a new 15 ml tube. Carefully add 3 ml buffer S3(0.88M Sucrose, 0.5mM MgC12) to the bottom of the tube and keep the two layers cleanly separated. Centrifuge at 4000 rpm for 10 min at 4℃ and discard the supernatant. Resuspend with 100 ul buffer S3, check pure isolated nucleoli under a microscope and store at -80℃.
All libraries were prepared for sequencing using standard Illumina protocols

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

HiSeq X Ten

Description

for more detials see DataFiles.README

Data processing

Library strategy: WGS
DNA sequencing data processing. We prepared HeLa cells and U2OS cells, and then obtained the HeLa cells whole genome sequencing (WGS) data, HeLa nucleolus sequencing data, HeLa nucleolus sequencing data before and after ActD treatment and U2OS WGS data with 150 bp paired-end sequencing on Illumina XTen platform. We processed these DNA sequencing data using the normal pipeline: bowtie2 mapping, samtools sorting, and then removing the duplicates with Picard. To ensure the mapping quality, especially in nucleolus data set, we kept the mapped reads only with a MAPQ value higher than 20.
In situ Hi-C and nHi-C data processing. For all Hi-C data sets, we used the following analysis pipeline. We first trimmed each FASTQ reads to 36 bp, and then mapped reads with bowtie2. We also added the rDNA 43kb repeat unit sequence as an artificial chromosome in genome reference. After reads mapping, we only kept the reads with MAPQ higher than 20, and we filtered the illegal interactions, which contains no restriction enzyme site within a downstream 500 bp region. We then filtered out the interactions where two paired reads map to the same restriction enzyme fragment. The in situ Hi-C and nHi-C data set are processed with the same pipeline. We used the ICE normalization method35 to clean the matrix for all in situ Hi-C matrixes.
Genome_build: hg19
Supplementary_files_format_and_content: matrix file are proformed with Hi-C data processing pipeline use Python script. And each matrix can be loaded with "," as separater

Submission date

Nov 17, 2016

Last update date

Feb 01, 2021

Contact name

Howard MENG

E-mail(s)

[email protected]

Organization name

Peking University

Department

School of Life Sciences