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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 07, 2024 |
| Title |
Bulk, HEK293T, Mock, rep2 |
| Sample type |
SRA |
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| Source name |
HEK293T
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
genotype: wildtype
treatment: Mock
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| Treatment protocol |
Stereotaxic injections were performed on mice with AAV viruses; HEK293T cells were transduced;
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| 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.
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| 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)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| 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
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| Submission date |
Aug 15, 2023 |
| Last update date |
May 07, 2024 |
| Contact name |
Magdalena Justyna Koziol |
| E-mail(s) |
mjk@cibr.ac.cn
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| Organization name |
Chinese Institute for Brain Researach, CIBR
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| Street address |
Science Park Road
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| City |
Beijing |
| ZIP/Postal code |
102206 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE240863 |
Single cell discovery of m6A RNA modifications in the hippocampus |
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| Relations |
| BioSample |
SAMN36994085 |
| SRA |
SRX21372239 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7710950_20-Bulk_HEK293T_Mock_rep2_Venn.cims.txt.gz |
205.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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