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Sample GSM5680710

Query DataSets for GSM5680710

Status

Public on Aug 01, 2022

Title

m6A_IP_WT_rep1

Sample type

SRA

Source name

mouse immune cells

Organism

Mus musculus

Characteristics

cell type: M1-type macrophages
strain: C57BL/6
genotype: WT
antibody: m6A (Synaptic, 202003)

Treatment protocol

M1-type BMDMs were purified from WT or Ythdf2 cKO mice.

Growth protocol

Bone marrow was extracted from the femurs and tibias of WT or Ythdf2 cKO mice. The whole bone marrow cells were flushed out and filtered using a 70 µm Cell Strainer. After centrifugation, red blood cells were lysed. The resultant bone marrow cells were resuspended in RPMI-1640 (Gibco) supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (Gibco), 50 μM 2-Mercaptoethanol (Sigma) in the presence of 20 ng/mL M-CSF (PeproTech) for 7 days. BMDMs were stimulated with 100 ng/ml LPS (Sigma) plus 20 ng/ml IFN-γ (PeproTech) for 24 h to obtain M1 type macrophages.

Extracted molecule

polyA RNA

Extraction protocol

For m6A-seq, total RNA was isolated by TRIzol reagent. Polyadenylated RNA was further enriched from total RNA by using Dynabeads® mRNA Purification Kit (Invitrogen). mRNA samples were fragmented into ~100-nucleotide-long fragments with RNA fragmentation reagents (Invitrogen). Fragmented mRNA (5 μg mRNA) was used for m6A immunoprecipitation (m6A-IP) using an m6A antibody (Synaptic, 202003) following the standard protocol of the Magna MeRIP m6A Kit (Merck Millipore). For Ythdf2 RIP-seq, fifty million BMDMs were harvested and washed twice with cold PBS, and the cell pellet was lysed with two volumes of lysis buffer [10 mM HEPES pH 7.6, 150 mM KCl, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 Protease Inhibitor cocktail (Thermo Fisher Scientific), and 400 U/mL SUPERase-In RNase Inhibitor (Thermo Fisher Scientific)]. The lysate was incubated on ice for 5 min and centrifuged for 15 min to clear the lysate. One-tenth volume of cell lysate was saved as input. The rest of the cell lysate was incubated with 5 μg anti-YTHDF2 (MBL, Cat No. RN123PW) that was pre-coupled with Protein A magnetic beads (Invitrogen, Cat: 10001D) at 4℃ for 2h with gentle rotation. Afterward, the beads were washed five times with 1 ml ice-cold washing buffer (50 mM HEPES pH 7.6, 200 mM NaCl, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, and 200 U/mL RNase inhibitor). Immunoprecipitated samples were subjected to Proteinase K digestion in wash buffer supplemented with 1% SDS and 2 mg/mL Proteinase K (Thermo Fisher Scientific) incubated with shaking at 1200 rpm at 55℃ for 1 h. Total RNA was extracted from both input and immunoprecipitated RNA by adding 5 volumes of TRIzol reagent, followed by Direct-zol RNA Miniprep (Zymo Research).
For m6A-seq,RNA was enriched through RNA Clean & Concentration- 5 (Zymo Research) for library generation with a KAPA RNA HyperPrep Kit (Roche). Sequencing was performed at the City of Hope Genomics Facility on an Illumina NovaSeq 6000 machine with 100-bp paired-end reads (PE100). For Ythdf2 RIP-seq, cDNA library generation was produced with a KAPA RNA HyperPrep Kit (Roche) and sequenced on Illumina NovaSeq 6000 platform.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

WT m6A exomepeak2.xls.txt
Project_COHP_44408_1_KBMRS

Data processing

Library strategy: m6a-seq
Sequencing reads were mapped to the mouse genome using HISAT2 (Version: v101)
Mapped reads of IP and input libraries were provided for R package exomePeak
The m6A binding motif was analyzed by HOMER.
Called peaks were annotated by intersection with gene architecture using R package ChIPseeker
Peak calling results with IP and input libraries were generated by R package RIPSeeker
Called peaks were annotated by intersection with gene and transcript architecture using ChIPpeakAnno
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample, peak text files

Submission date

Nov 08, 2021

Last update date

Aug 01, 2022

Contact name

Shoubao Ma

E-mail(s)

shma@coh.org

Phone

6262182182

Organization name

City of Hope