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Sample GSM7863661

Query DataSets for GSM7863661

Status

Public on Aug 26, 2024

Title

Curlcake_m5C_rep1_ivt

Sample type

SRA

Source name

synthetic construct

Organism

synthetic construct

Characteristics

tissue: synthetic construct

Extracted molecule

polyA RNA

Extraction protocol

In vitro constructs were artifically poly-A-tailed using PAP (NEB, M0276S), and cleaned up using the RNEasy MinElute kit (Quiagen, 74204) for curlcake constructs and Zymo Clean and Concentrator kit (Zymo Resarch, R1013). Concentrations were determined using a Qubit 2.0 Fluorometer. For libraries that were not generated as part of this work please refer to the respective publications
Direct RNA-seqeuncing libraries were generated using the SQK-RNA002 according to the manufacturer's protocol (version : DRS_9080_v2_revR_14Aug2019-minion). For curlcakes a final library of 800ng war generated (200ng per curlcake construct) while 750ng for the UNM-S libraries was used (250ng per riboswitch). The reverse transcription step was peformed using SuperScript III R(Thermo Fisher Scientific, 18080044) in the case of ac4C, m5U-containing curlcake constructs and UNM-S, and Superscript IV (Thermo Fisher Scientific, 18090010) in the case of m1Ψ. For libraries that were not generated as part of this work please refer to the respective publications.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

MinION

Data processing

Processing of raw fast5 files was peformed using the MasterOfPores nextflow pipeline. Reads were basecalled using guppy (v6.0.6) with each of the three tested basecalling models (default, ivt and sup).
Alignments to both reference genome ( -uf -ax splice -k14) and transcriptome (-ax map-ont -k14) were performed using minimap2 (v2.17). Highly modified synthetic constructs and the corresponding unmodified control were mapped using graphmap (v0.5.2) with sensitive settings (--rebuild-index -v 1 --double-index --mapq -1 -x sensitive -z 1 -K fastq --min-read-len 0 -A 7 -k 5)
To extract error signature on a per-nucleotide basis, EpinanoRMS (v1.1) was run. For in vivo m6A analyisis, pairwise-comparisons between wildtype and knockout samples were performed using eligos2(v2.1.0). Cross-species comparison of read accuacies was performed on reads uniquely mapped to the reference transcriptome (-F 3840) with a mapping quality > 15 (-q 15). To balance reads for strongly varying expression profiles at most the 10 longest reads for each transcript were extracted (bam2select.py). Read identis were extracted using identity_density.py
Assembly: Reference files include curlcake_constructs_EcoRV_BamHI_digestion.fasta, riboswitches.fa and 2021-8_eGFP_mRNA.v2.fa for curlcakes, riboswitches and the syntehtic eGFP vaccine, respectively. We used the following genomic reference for H. sapiens : Homo_sapiens.GRCh38.dna_sm.primary_assembly.fa. And transcriptomic references Homo_sapiens.GRCh38.cdna.all.fa, Mus_musculus.GRCm39.cdna.all.fa, xlaevisMRNA.fasta, Saccharomyces_cerevisiae.R64-1-1.cdna.all.fa and Arabidopsis_thaliana.TAIR10.cdna.all.fa for H. sapiens, M. musculus, X. laevis, S. cervisiae and A. thaliana, respecitvely.
Supplementary files format and content: Files ending in <model>_per_position.csv, contain EpinanoRMS results on a per position basis. Files ending in <model>_per_5mer.tsv contain EpinanoRMS results on a 5-mer basis generated by converting per position results using epinano_to_kmer.R.
Supplementary files format and content: Files ending in _eligos2.txt contain results generated by eligos2 for pairwise comparisons which were already filtered to remove non-A-positions and homopolymer sequences.
Supplementary files format and content: Files starting with <species>.tsv contain read accuracies per aligned read (rows) for all three models (column 1).

Submission date

Oct 24, 2023

Last update date

Aug 26, 2024

Contact name

Gregor Diensthuber

E-mail(s)

gregordiensthuber@gmail.com

Phone

+34656764194

Organization name

Center for Genomic Regulation (CRG)