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Sample GSM3142555

Query DataSets for GSM3142555

Status

Public on Jun 13, 2018

Title

SMART_seq2_R23E10_Cell_25

Sample type

SRA

Source name

SMART_seq2_R23E10_Cell

Organism

Drosophila melanogaster

Characteristics

age: 0-7 Days
genotype/variation: R23E10-Gal4 x UAS-CD8::GFP
tissue: brain

Treatment protocol

Flies were grown on molasses based flyfood at 25'C with a 12hour/12hour day night cycle

Extracted molecule

polyA RNA

Extraction protocol

Adult flies were anesthetised by placing them in ice cold glass dishes, heads were removed and brains were dissected removing any cuticle, retina and trachea. Dissected brains were placed in ice cold PBS. When 40 brains were collected, brains were dissociated at 25'C for 2 hours in a mix of Collagenase (75µl @ 100mg/ml) and Dispase (50µl @ 3mg/ml), the sample was pipette mixed every 15 minutes. Cells washed in PBS twice centrifuging at 800 xg) and resuspended in 400µl PBS. A single cell suspension was obtained by filtering with an 11µm cell strainer. Cells were sorted into single wells containing SMART-seq lysis buffer.
SMART-Seq

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

processed data file:
R23E10_SMART-seq2_DGEM.tsv.gz

Data processing

The 10X samples were each processed (alignment, barcode assignment and UMI counting) with Cell Ranger (version 2.0.0) count pipeline. All 10X samples were then merged with Cell Ranger (version 2.0.0) aggr pipeline.
The Drop-seq samples were each processed (alignment, barcode assignment and UMI counting) with the Drop-seq tools (version 1.12) developped by Jim Nemesh from McCarroll lab. Gene expression matrices where outer joined.
The reads from CEL-seq2 samples were first cleaned for adapters using fastq-mcf (Aronesty, 2011) and a list of sequencing primers. Multiplexed CEL-seq2 cells were demultiplexed with custom scripts utilizing pysam (Li et al., 2009). Cleaned reads were then mapped to the 3rd 2017 FlyBase release (D. melanogaster r6.16) genome using STAR with default parameters. Reads were counted per gene using featureCounts.
The reads from SMART-seq2 and Adapted SMART-seq2 samples were mapped using STAR (2.5.1) and the expression matrices were generated using featureCounts from Subread 1.5.1.
For the ATAC samples the raw reads were first filtered and scanned for adapters using fastq‐mcf from the ea‐utils r819 package. Read quality was then checked using FastQC 0.11.4. The reads were mapped to the Drosophila genome (dm6) using STAR 2.5.1. The outputted BAM file from STAR was then indexedand sorted using Samtools 1.2. Using genomeCoverageBed from Bedtools 2.23.0, 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)
Supplementary_files_format_and_content: 10X DGRP-551 + w1118 high quality dataset (57k) raw UMI count matrix stored in compressed Market Exchange Format (MEX). It also contains TSV files with genes and barcode sequences corresponding to row and column indices, respectively
10X DGRP-551 + w1118 large dataset (157k) raw UMI count matrix stored in compressed Market Exchange Format (MEX). It also contains TSV files with genes and barcode sequences corresponding to row and column indices, respectively
CEL-seq2 FAC-sorted R23E10 neurons raw count matrix
SMART-seq2 FAC-sorted R23E10 neurons raw count matrix
Adapted SMART-seq2 FAC-sorted R23E10 neurons raw count matrix
Drop-seq DGRP-551 dataset raw UMI matrix
ATAC-seq bigWig files

Submission date

May 15, 2018

Last update date

Jun 13, 2018

Contact name

Stein Aerts

E-mail(s)

[email protected]

Organization name

KULeuven/VIB

Department

Center for Brain & Disease Research