MassIVE MSV000094375

Partial Public PXD050870

Single-cell m6A profiling in the mouse brain uncovers cell type-specific RNA methylomes and age-dependent differential methylation

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Description

Ten micrograms of each sample were reduced and alkylated followed by trypsin digestion using a suspension trap (S-trap). An SPQC was made by combining equal volumes of each sample. After lyophilization and reconstitution of the sample in 20 uL, 1-2 uL of each was analyzed by LC-MS/MS using a 30 min gradient on a Thermo Vanquish Neo coupled to a Thermo Orbitrap Astral via a Nanospray Flex ionization source. Briefly, the sample was first trapped on a PepMap C18 0.3 mm x 5 mm trapping column (50 ul/min at 99.9/0.1 v/v water/acetonitrile), followed by analytical separation using a 1.5 um 150um ID x 8cm (PepSep) column with a 20 min gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 500 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Orbitrap Astral mass spectrometer was performed in a data-independent acquisition (DIA) mode of acquisition with a r=240,000 (@ m/z 200) full MS scan every 0.6s from m/z 380-980 with an AGC target of 500%. DIA MS/MS scans were acquired in the Astral analyzer using fixed windows of 4 m/z from m/z 380, target AGC of 500% and max fill time of 6 ms. HCD collision energy setting of 27% was used for all MS2 scans. Raw MS data was demultiplexed and converted to *.htrms format using HTRMS converter and processed in Spectronaut 18 (Biognosys). A spectral library was built using direct-DIA searches which used a Mus muculus database, downloaded from Uniprot on 4/08/23, and appended the transgene APOBEC1-YTH as well as contaminant sequences using FragPipe. Search settings included N-terminal trypsin/P specificity up to 2 missed cleavages; peptide length from 7-52 amino acids with the following modifications: acetyl (N-term), carbamidomethyl(Cys) and oxidation (M). For DIA analysis, default extraction, calibration, identification and protein inference settings were used. Normalization, imputation and protein quantification was performed using an in-house script. [doi:10.25345/C5Z892S0H] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: m6A (or N6-methyladenosine) ; DART-seq ; RNA modifications ; Transgenic Mouse Characterization

Contact

Principal Investigators:
(in alphabetical order) 		Kate Meyer, Duke University, USA
Submitting User: mviolette
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