NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM6211243 Query DataSets for GSM6211243
Status Public on Jun 15, 2022
Title wing_disc_control_rep1_multiome_atac
Sample type SRA
 
Source name wing imaginal disc
Organism Drosophila melanogaster
Characteristics tissue: wing imaginal disc
developmental stage: thrid instar larvae
condition: control
genotype: w1118; +; rn-Gal4, UASeiger, tub-Gal80ts/TM6B
treatment: none
Treatment protocol Overexpression of the TNF ligand egr transgene was induced by shifting the temperature to 30°C for 24h at day 7 after egg deposition
Growth protocol Flies were raised at 18°C at a density of 50 larvae/vial
Extracted molecule genomic DNA
Extraction protocol Control and wounded wing discs were dissected and transferred to a tube containing ice cold PBS. PBS was removed by centrifugation, tissues were flash frozen in liquid nitrogen and stored at -80°C. The following procedure was followed to extract the nuclei from the wing discs:  resuspension in 500 µl nuclei lysis buffer comprising 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40, 0.01% Digitonin, 1% BSA, 1 mM dithiothreitol and 1U/µl RNasin ribonuclease inhibitor (Promega) in nuclease-free water, incubation 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 added 1 ml of wash buffer composed of 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween 20, 1% BSA, 1 mM dithiothreitol and 1U/µl RNasin ribonuclease inhibitor in nuclease-free water. Nuclei were pelleted by centrifugation at 800 g for 5 min at 4°C and resuspended in a 1x nuclei buffer (10x Genomics) supplemented with 1 mM dithiothreitol and 1U/µl RNasin ribonuclease inhibitor. Nuclei suspensions were passed through a 40 µm Flowmi filter (VWR Bel-Art SP Scienceware). Nuclei concentration was assessed using the LUNA-FL Dual Fluorescence Cell Counter.
Single-cell libraries were generated using the 10x Chromium Single-Cell Instrument and NextGEM Single Cell Multiome ATAC + Gene Expression kit (10x Genomics) according to the manufacturer’s protocol. In brief, the single nuclei of wing discs were incubated for 60 min at 37°C with a transposase that fragments the DNA in open regions of the chromatin and adds adapter sequences to the ends of the DNA fragments. After generation of nanolitre-scale gel bead-in-emulsions (GEMs), GEMs were incubated in a C1000 Touch Thermal Cycler (Bio-Rad) under the following program: 37°C for 45 min, 25°C for 30 min and hold at 4°C. Incubation of the GEMs produced 10x barcoded DNA from the transposed DNA (for ATAC) and 10x barcoded, full-length cDNA from poly-adenylated mRNA (for GEX). This was followed by a quenching step that stopped the reaction. After quenching, single-cell droplets were broken and the transposed DNA and full-length cDNA were isolated using Cleanup Mix containing Silane Dynabeads. To fill gaps and to generate sufficient mass for library construction,  the transposed DNA and cDNA were amplified via PCR: 72°C for 5 min;  98°C for 3 min; 7 cycles of 98°C for 20 s, 63°C for 30 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. The pre-amplified product was used as input for both ATAC library construction and cDNA amplification for gene expression library construction. Illumina P7 sequence and a sample index were added to the single-strand DNA during ATAC library construction via PCR: 98°C for 45 s; 7-9 cycles of 98°C for 20 s, 67°C for 30 s, 72°C for 20 s; 72°C for 1 min; and hold at 4°C. The sequencing-ready ATAC library was cleaned up with SPRIselect beads (Beckman Coulter). Barcoded, full-length pre-amplified cDNA was further amplified via PCR: 98°C for 3 min; 6-9 cycles of 98°C for 15 s, 63°C for 20 s, 72°C for 1 min; 72°C for 1 min; and hold at 4°C. Subsequently, the amplified cDNA was fragmented, end-repaired, A-tailed and index adaptor ligated, with SPRIselect cleanup in between steps. The final gene expression library was amplified by PCR: 98°C for 45 s; 5-16 cycles of 98°C for 20 s, 54°C for 30 s, 72°C for 20 s. 72°C for 1 min; and hold at 4°C. The sequencing-ready GEX library was cleaned up with SPRIselect beads.
 
Library strategy ATAC-seq
Library source genomic single cell
Library selection other
Instrument model Illumina NovaSeq 6000
 
Description REW__e37570__709b79__Dros_rotundGAL4_GAL80ts_ctrl_10x_multi_s2.barcodes.tsv.gz
REW__e37570__709b79__Dros_rotundGAL4_GAL80ts_ctrl_10x_multi_s2.features.tsv.gz
REW__e37570__709b79__Dros_rotundGAL4_GAL80ts_ctrl_10x_multi_s2.matrix.mtx.gz
Data processing Sequencing data were processed with CellRangerARC (v1.0.1) with default parameters and FlyBase r6.35 reference genome
scRNA data was processed using the NextFlow pipeline VSN (Flerin et al., 2021). VSN processing includes the doublet filtering using scrublet (Wolock et al., 2019) and the correction for batch effects between the runs using Harmony (Korsunsky et al., 2019). We used the following filtering parameters : minimum of 200 detected genes per cell, maximum of 15% signal of mitochondrial origin, minimum of 3 cells expressing any targeted genes.
scATAC data was processed using cisTopic (Bravo González-Blas et al., 2019) using 76 topics. scATAC count data were generated from a consensus set of 41,387 peaks based on peak calling from pseudobulk aggregate from scRNA clusters. We used the following filtering parameters : minimum number of fragment per cell of log10(3.5), minimum fraction of reads in CPS of 60%, minimum TSS enrichment of 2.
Assembly: dm6
Supplementary files format and content: Consensus peak set for scATAC is available as consensus_peak_set_atac.bed
Supplementary files format and content: CellRangerARC output for each multiome sample (control and wounded) comprising both ATAC and GEX features are present as TSV (barcode and features) and matrix (raw counts) format.
 
Submission date Jun 02, 2022
Last update date Jun 15, 2022
Contact name Stein Aerts
Organization name VIB-KU Leuven
Department Department of Human Genetics / VIB Center for Brain and Disease Research
Lab Laboratory of Computational Biology
Street address Herestraat 49
City Leuven
State/province Flemish-Brabant
ZIP/Postal code 3000
Country Belgium
 
Platform ID GPL25244
Series (2)
GSE205387 Shared enhancer gene regulatory networks between wound and oncogenic programs [Multiome]
GSE205401 Shared enhancer gene regulatory networks between wound and oncogenic programs
Relations
BioSample SAMN28843076
SRA SRX15576587

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

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap