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Sample GSM4984044

Query DataSets for GSM4984044

Status

Public on Aug 10, 2021

Title

pupa72h_brain_DGRP-409-502_sample2

Sample type

SRA

Source name

Pupa Brain

Organism

Drosophila melanogaster

Characteristics

tissue: Brain
developmental stage: Pupa
age: 72 hours
genotype: DGRP-409-502

Extracted molecule

genomic DNA

Extraction protocol

D. melanogaster adult brains were dissected and transferred to a tube containing 100 µl ice cold DPBS solution. After centrifugation at 800 g for 5 min, the supernatant was replaced by 500 µl nuclei lysis buffer composed of 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40, 0.01% Digitonin, and 1% BSA, in Nuclease-free water. The following procedure was followed to extract the nuclei from the brain tissue: incubation in nuclei lysis buffer on ice for 5 min, transfer to a dounce tissue grinder tube (Merck), 25 strokes with pestle A, incubation on ice for 10 min, 25 strokes with pestle B. The lysis was stopped by adding 1 ml of wash buffer composed of 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20 and 1% BSA, in Nuclease-free water. Nuclei were pelleted by centrifugation at 800 g for 5 min at 4°C and resuspended in 1x Nuclei Buffer (10x Genomics). Nuclei suspensions were passed through a 40 µM Flowmi filter (VWR Bel-Art SP Scienceware). Nuclei concentration was assessed by the LUNA-FL Dual Fluorescence Cell Counter.
10x Genomics CHROMIUM Single Cell ATAC Library & Gel Bead Kit v1

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Data processing

The 10X samples were each processed (alignment, barcode assignment and UMI counting) with Cell Ranger ATAC (version 1.2.0) count pipeline.
For the ATAC samples the raw reads were first filtered and scanned for adapters using fastq‐mcf from the ea‐utils package. Read quality was then checked using FastQC. The reads were mapped to the Drosophila genome (dm6) using STAR. The outputted BAM file from STAR was then indexedand sorted using SAMtools. Using genomeCoverageBed from BedTools, the BAM file was converted to a BedGraph file. For fast, visualization in the IGV GenomeBrowser, the BedGraph file was further processed to a BigWig file using bedGraphtoBigWig.
Genome_build: dm6 (dmel_r6.16_FB2017_03)
81 bigwigs are attached based on samples belonging to 10X_ADULT (adult brain) and to 10X_PUPA_72H
10X_ADULT and 10X_PUPA_72H samples were subset based on cell type and merged to acquire cell type specific bam files
bam files were then deduplicated, black listed regions removed and RPGC normalised to acquire bigwig files

Submission date

Dec 22, 2020

Last update date

Aug 10, 2021

Contact name

Stein Aerts

E-mail(s)

[email protected]

Organization name

KULeuven/VIB

Department

Center for Brain & Disease Research