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Sample GSM4145192

Query DataSets for GSM4145192

Status

Public on Oct 09, 2020

Title

HeLa cells, WT (sg-Control) input rep 2

Sample type

SRA

Source name

HeLa cells

Organism

Homo sapiens

Characteristics

genotype: WT
cell line: HeLa
treatment: transfected with GCN4-GGS6-dCas13b, scFv-GGS6-FTO-GB1 and sg-Control for overall 48 hours.
rip antibody: none

Treatment protocol

The co-transfection ratio of GCN4-GGS6-dCas13b, scFv-effector protein-GB1 and sgRNA-expressing plasmid is 1:1:2. For m6A-seq, cells were transfeced by identical plasmids for overall 48 hours. For RNA lifetime profiling, cells were transfected by identical plasmids for overall 48 hours. And actinomycin D was added to 5 ug/mL at 6 hours, 3 hours and 0 hour before cell scraping collection.

Growth protocol

HeLa (ATCC) cells were maintained using DMEM (high glucose) supplemented with 10% fetal bovine serum, and 1% penicillin/streptomycin.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was isolated by TRIzol reagent (Invitrogen). PolyA-RNA was isolated by two rounds of ployA selection with Oligo d(T)25 Magnetic Beads (NEB, S1419S) from total RNA and followed by additional TURBO DNase treatment. mRNA were purified by RNA Clean & Concentrator Kits and then fragmented to about 100 nt using RNA Fragmentation Reagents (Invitrogen, AM8740). The fragmented mRNA were purified by using RNA Clean & Concentrator Kits. For m6A-seq, while 100 ng mRNA was saved as input sample, the rest mRNA was used for m6A-immunoprecipitation. Briefly, nearly 5 ug mRNA was incubated with m6A antibody (Synaptic Systems, 202 003) and diluted into 500 uL IP buffer (150 mM NaCl, 0.1% Igepal CA-630, 10 mM Tris, pH 7.4, 100 U RNase inhibitor). The mixture was rotated at 4°C for 2 hours, following Dynabeads® Protein A (Invitrogen, 10002D) was added into the solution and rotated for another 2 hours at 4 °C. After four times washing by IP buffer, the m6A IP portion was eluted twice by 100 uL m6A-elute buffer (IP buffer, 6.7mM m6A, 30 U RNase inhibitor) with incubating and shaking at 4°C for 1 hour. Finally, m6A IP mRNA was recovered by RNA Clean & Concentrator Kits and the RNA concentration was measured with Qubit® RNA HS Assay Kit (Invitrogen, Q32855).
Sequencing libraries of input and ip samples were prepared using NEBNext Ultra II RNA Library Prep Kit.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Description

RNA-seq
m6a_peak_wildtype_sgControl_rep2.tsv.gz

Data processing

Adapter sequences were trimmed using Cutadapt(2.5) program.
Potential ribosomal RNA contamination were eliminated by using Bowtie(1.2.2) program to align reads to reference ribosomal RNA sequences.
Reads were mapped to GRCh38 by using STAR(STAR_2.5.0a) program. Only proper-pair and primary alingments were retained.
Fragment counts and FPKM of transcripts were counted by using custom scripts.
Lifetime were calculated by the comparison of the FPKM at different time points.
m6A enriched peaks were identified by comparing the normalized coverage of IP and Input.
Genome_build: GRCh38
Supplementary_files_format_and_content: Tab-delimited text files include the fragment count and FPKM values for all samples and RNA lifetime calculated by FPKM of different time points. Bed files include the peak regions on BED12 format.

Submission date

Oct 31, 2019

Last update date

Oct 09, 2020

Contact name

Zonggui Chen

E-mail(s)

chenzonggui@outlook.com

Organization name

Peking University

Department

Academy for Advanced Interdisciplinary Studies