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Sample GSM6265722

Query DataSets for GSM6265722

Status

Public on Dec 01, 2023

Title

Third instar CNS, gWT3

Sample type

SRA

Source name

central nervous system

Organism

Drosophila melanogaster

Characteristics

tissue: central nervous system
developmental stage: third instar larvae
genotype: w1118

Extracted molecule

genomic DNA

Extraction protocol

Third instar CNS were dissected in ice cold PBS. Samples 1-6: Tissues were crushed in lysis buffer (100 mM Tris-HCl pH9, 1 mM EDTA, 1% SDS), digested with RNAse A for 15 min at room temperature and 30 min at 65°C, before being incubated with 4 volumes of 3.2 M LiCl, 0.9 M KAc for 30 min at 4°C. After centrifugation at 12000 g for 20 min at 4°C, the supernatant was extracted with phenol-chloroform-isoamyl alcohol and DNA was precipitated with isopropanol. Samples 7-11:RNAs were extracted with Qiagen RNeasy Plus Micro kit and samples were converted to cDNA using Nugen’s Ovation RNA-Seq System.
Samples 1-6: libraries were prepared using a SMRTbell Express 2 Template prep kit. Samples 7-11: library preparation was performed using Kapa Biosystems library preparation kit.
Samples 1-6: SMRT-seq. Samples 7-11: RNA-seq

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Sequel

Description

WT_fusion_all.xlsx
Sample 3

Data processing

Illumina Hiseq2500 reads were trimmed using Trimmomatic and checked with FASTQC
Illumina reads were aligned to the Drosophila reference genome (dm6 Ensembl release 70) with Hisat2
Read counts were generated for each annotated gene using HTSeq-Count. RPKM (Reads Per Kilobase of exon per Megabase of library size) values were calculated using Cufflinks. Reads normalization, variance estimation and pair-wise differential expression analysis with multiple testing correction were conducted using the R Bioconductor DESeq2 package
SMRT-seq reads were aligned using pbmm2 tool (https://github.com/PacificBiosciences/pbmm2) on Drosophila genome (dm6 Ensembl realease 70)
6mA detection was performed on every samples and on merged ones with IpdSummary tool from KineticTools (http://github.com/PacificBiosciences/kineticsTools) applying following parameters: --identify m6A --numWorkers 16 --pvalue 0.01 --identifyMinCov 5 –methylFraction
Assembly: Drosophila genome (dm6 Ensembl release 70)
Supplementary files format and content: text file with RNA-seq expression
Supplementary files format and content: Excel file with 6mA detected localization and scores

Submission date

Jun 24, 2022

Last update date

Dec 01, 2023

Contact name

yoan renaud

E-mail(s)

yoan.renaud@uca.fr

Organization name

igred

Street address

place henri dunand