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Sample GSM7045807

Query DataSets for GSM7045807

Status

Public on Feb 16, 2023

Title

HeLa siControl Input rep2

Sample type

SRA

Source name

Uterus

Organism

Homo sapiens

Characteristics

tissue: Uterus
cell line: HeLa
cell type: epithelial cell
genotype: control knockdown
treatment: UV 254 nm crosslink

Treatment protocol

Irradiated twice with 400 mJ/cm2 at 254 nm in Stratalinker on ice.

Growth protocol

HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C

Extracted molecule

total RNA

Extraction protocol

After irradiation, cells were harvested with a scraper, transferred to a microtube, pelleted at 4000 rpm for 3 min at 4°C, and then lysed in lysis buffer (150 mM NaCl, 0.5% NP-40, 50 mM Tris-HCl (pH 7.5), 2 mM EDTA) with cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail and SUPERase In at 4°C for 1 hour. Subsequently, the lysis mixture was centrifuged at 17,000 g at 4°C for 30 min and the supernatant was carefully collected. The samples were then treated with RNase T1 (1 u/µL) at RT for 15 min and centrifuged to collect the supernatant, 10% of which was saved as input. Then YTHDF2 antibody-conjugated protein G beads prepared by incubating antibody and beads at 4°C for 6 hours were added into the samples and incubate overnight. After beads washing, 10 u/µl RNase T1 was added and incubated at RT for 8 minutes. After incubation, the beads were extensively washed and then resuspended in 50 µL of SDS-PAGE loading buffer and heated at 95°C for 5 min. The RNA was finally extracted by cutting and recovering the band of YTHDF2-RNA complex from the SDS-PAGE gel. Before library preparation, end-repair by PNK and ATP was performed for the input and IP RNAs. The
Library was prepared by NEBNext® Small RNA Library Prep kit following manufacturer's protocols.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Data processing

Low quality reads were filtered using ‘fastq_quality_filter’, and adapter were clipped using ‘fastx_clipper’, then adapter-free were collapsed to remove PCR duplicates by using ‘fastx_collapser’ and finally reads longer than 15 nt were retained for further analysis (http://hannonlab.cshl.edu/fastx_toolkit/). Reads from rRNA were removed.
The preprocessed reads were mapped to hg38 using bowtie with ‘-v 3 -m 10 -k 1 –best –strata’ parameters.
Genome_build: hg38
Supplementary_files_format_and_content: text files include read counts for each Sample

Submission date

Feb 15, 2023

Last update date

Feb 16, 2023

Contact name

Philip Cody He

E-mail(s)

[email protected]

Organization name

University of Chicago

Street address

929 E 57th Street