|
| Status |
Public on Apr 26, 2022 |
| Title |
Mettl3_KO_Soma_KCl_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Hippocampal Neurons
|
| Organism |
Mus musculus |
| Characteristics |
strain: Mettl3Flox/Flox
genotype: Mettl3-/-
compartment: Soma
treatment: Treated with KCl for 2 h.
library type: RNAseq
|
| Treatment protocol |
Cells were transduced with AAV particles expressing Cre-P2A-dTomato at DIV7 (50,000 vg/cell). Cells were treated with 1 µM tetrodotoxin and 50 µM D-AP5 for 16 hours at DIV14. For treated cells, KCl was added to a final concentration of 55 mM for 2 hours prior to RNA extraction.
|
| Growth protocol |
Primary hippocampal neurons isolated from neonate mouse were plated on PET microporous membranes (Millipore) coated with poly-D-lysine and laminin and maintained in growth media (Neurobasal A, B27, 0.6mM Glutamax) for 15 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Soma and neurites fractions were respectively isolated from the top and bottom of the microporous membrane using a cell scraper. RNA extraction was performed with the Qiagen RNeasy Mini plus kit.
For all samples, 50ng of total RNA was used for library preparation with the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina. For DART-seq samples, 50ng of total RNA was incubated with 250ng of recombinant APOBEC1-YTH protein for 6 h at 37C prior to library preparation.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
featureCounts.output.txt
DESeq2_KCl_treated_neurite_enrichment.txt
|
| Data processing |
Adapters were trimmed from the 3' end of reads using Flexbar with the following options: --adapter-trim-end LEFT --adapter-revcomp ON --adapter-revcomp-end RIGHT --htrim-left GT --htrim-right CA --htrim-min-length 3 --htrim-max-length 5 --htrim-max-first --htrim-adapter --min-read-length 2
Reads were mapped to mouse genome (mm10) and duplicates marked using STAR v2.7.7a and the following options: ' --runMode alignReads --outSAMtype BAM SortedByCoordinate --readFilesCommand zcat --outFilterMismatchNmax 30 --outSAMattributes All --outFilterScoreMinOverLread 0.25 --outFilterMatchNminOverLread 0.25', followed by running STAR with the following options: '--runMode inputAlignmentsFromBAM --bamRemoveDuplicatesType UniqueIdentical'
For RNAseq analysis, raw gene counts were calculated using featureCounts v2.0.1 with the following options: "-M -p -t exon -g gene_id". Neurite enrichment was calculated with DESeq2.
For DART-seq, bam files were parsed to build a matrix of coverage at each position in the transcriptome for each library. C-to-U mutations in WT cells were compared to background editing in Mettl3-/- libraries. m6A sites were called as those found in a RAC motif, with a at least 3 mutations, 50 covering reads, 1-95% C-to-U editing, more than 1.5-fold editing and foun in at least 2 biological replicates.
To compare editing levels across compartments in DART-seq experiments, the cummulative editing rates in each transcript was fitted in a binomial generalized linear model to obtain log2FoldChange and P-values (Wald-test).
Genome_build: mm10
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample.
Supplementary_files_format_and_content: Result of DESeq2 analysis for enrichment in neurites over soma for under untreated or KCl-treated conditions.
Supplementary_files_format_and_content: For DART-seq: bed files with position of m6A sites identified under each condition.
Supplementary_files_format_and_content: For DART-seq: Tab separated table containing the differential editing analysis in Soma and neurites for all transcript with mapped m6A sites.
|
| |
|
| Submission date |
Mar 30, 2021 |
| Last update date |
Nov 03, 2022 |
| Contact name |
Kate Meyer |
| E-mail(s) |
kate.meyer@duke.edu
|
| Phone |
9196849562
|
| Organization name |
Duke University School of Medicine
|
| Department |
Biochemistry
|
| Street address |
307 Research Drive Box 3711
|
| City |
D |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE171199 |
m6A and YTHDF proteins control the subcellular localization of select neuronal mRNAs [RNA-seq and DART-seq] |
| GSE194208 |
m6A and YTHDF proteins control the subcellular localization of select neuronal mRNAs |
|
| Relations |
| BioSample |
SAMN18543142 |
| SRA |
SRX10484605 |
