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Sample GSM5220671 Query DataSets for GSM5220671
Status Public on Apr 26, 2022
Title Mettl3_KO_Neurite_Ctrl_rep2
Sample type SRA
 
Source name Hippocampal Neurons
Organism Mus musculus
Characteristics strain: Mettl3Flox/Flox
genotype: Mettl3-/-
compartment: Neurite
treatment: untreated
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_untreated_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 SAMN18543131
SRA SRX10484616

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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