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Sample GSM7710933

Query DataSets for GSM7710933

Status

Public on May 07, 2024

Title

Hippocampus, m6A RIP-input, rep2

Sample type

SRA

Source name

hippocampus

Organism

Mus musculus

Characteristics

tissue: hippocampus
strain: C57BL/6J
genotype: wildtype
treatment: m6A RIP input control

Treatment protocol

Stereotaxic injections were performed on mice with AAV viruses; HEK293T cells were transduced;

Growth protocol

Mice: Hippocampi were harvested from 3 month old mice. Animals were maintained and processed in accordance with CIBR guidelines. All experimental methods were approved and followed all regulations of the Welfare and Ethics Review Committee for Laboratory Animals. C57BL/6J mice were originally purchase from the Jackson Laboratory, but were made available through CIBR’s animal facility. For HEK293T cells: HEK293T cells were purchased from Beijing Xiehe Cell Bank and cultured at 37ºC with 5% CO2 in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS and 1% penicillin/streptomycin.

Extracted molecule

total RNA

Extraction protocol

HEK293T cells were collected and RNA was extracted for downstream processing; For mouse hippocamus collections, hippocampi were dissected 2 weeks after AAV injectsions and RNA was extracted; for single cell analysis, cells were dissociated 2 weeks after AAV injections with using the Adult Brain Dissociation Kit (Miltenyi Biotex no. 130-107-677); for m6A-RIP, a pulldown was performed on RNA using the m6A antibody (Synaptic Systems, 202011), and a matching control input sample was keept.
for any bulk RNA seq: Sequencing libraries were generated from 1-10ng of total RNA from each replicate using the Single Cell Full Length mRNA kit and TruePrep® DNA Library Prep Kit V2 (Illumina), according to the manufacturer’s instructions. For single cell sequencing: Briefly, after hippocampi dissociation, cells were FACS sorted and EGFP positive were collected in ice-cold PBS containing 0.5% BSA. Per sample, 30,000 cells were loaded into a Rhapsody Cartridge (BD) and processed following the manufacturer’s instructions. Subsequently, single cell RNA sequencing libraries were prepared using the Rhapsody WTA kit (BD). For m6A-RIP: SMARTer Stranded Total RNA-Seq Kit v2 (Takara)
for single cells: Rhapsody WTA kit (BD); for bulk: TruePrep® DNA Library Prep Kit V2 (Illumina); for m6-RIP: SMARTer Stranded Total RNA-Seq Kit v2 (Takara)

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

02_WT_rep2_summits.bed

Data processing

For m6A-RIP: We required a minimum quality score of 5 at reads positions. adapters were removed using the software Cutadapt. Additionally, the first 10 nucleotides at the 5’ terminus were eliminated due to the presence of potentially low-quality nucleotides. Mapping and alignment were used by Hisat2, followed by peak calling using Macs2. To ensure reads unambiguously mapped in the genome, we further mapped the reads by bowtie2 alignment programs (The mapping parameters for bowtie2: -k 4, with other parameters in default setting.
For bulk-RNA-seq and m6A identification: Fastq files were filtered for low quality reads and low quality bases were trimmed from the ends of the reads. Bwa was used to align the resulting reads. To identify C-to-T and m6A sites, we used an approach as described by Meyer et al. (Meyer, 2019).
For single cell RNA-seq and m6A identification: Filtered barcode matrices obtained from E-YTH and E-YTHmut single cell data were processed by Seurat. The software Bullseye was used to identify m6A sites in single cell sequencing data (Tegowski et al., 2022).
For single cell RNA-seq matrix files were generated
cims files were generated
Assembly: Mouse: mm39; HEK293T: hg38
Supplementary files format and content: For m6A-RIP: m6A peaks
Supplementary files format and content: For bulk-RNA seq: cims files
Supplementary files format and content: For single cell RNA-seq: matrix files
Library strategy: m6A RIP-Seq

Submission date

Aug 15, 2023

Last update date

May 07, 2024

Contact name

Magdalena Justyna Koziol

E-mail(s)

mjk@cibr.ac.cn

Organization name

Chinese Institute for Brain Researach, CIBR

Street address

Science Park Road