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Sample GSM5421462

Query DataSets for GSM5421462

Status

Public on Feb 20, 2023

Title

sorted astrocytes 86E01 d5 rep2

Sample type

SRA

Source name

astrocytes

Organism

Drosophila melanogaster

Characteristics

age: d5
driver line: 86E01
sorting: sorted cells

Treatment protocol

For sorted cells: Brains from young (5 days old) and old (70 days old) females were dissected, collected into a tube with cold Schneider’s medium (SM) containing 1% BSA and washed twice. Next, we added 200 μL of dissociating solution, which included 2 mg/mL collagenase I (Sigma-Aldrich, SCR103) and 20 μg/mL DNase I (Worthington, Cat#LS002006) in SM containing 1% BSA with 45 μM actinomycin D to block new transcription during enzyme digestion. Brains were dissociated at 25°C using a shaker at 500 rpm for 1 hour and vigorous pipetting every 20 min. Dissociated tissue was filtered through 100 μm cell strainers and sorted at cell sorting facility of the University of Pennsylvania. Dead cells were excluded by staining with 4,6-diamidino-2-phenylindole (DAPI). GFP+ cells gates were set according to the fluorescence profile of GFP- brain tissue. Desired glia populations were directly sorted into TRIzol for RNA extraction.

Growth protocol

Stocks were maintained and experiments conducted at 25˚C and 50% humidity on a 12-hour light/dark cycle using standard Bloomington Drosophila medium (Nutri-Fly).

Extracted molecule

polyA RNA

Extraction protocol

RNA was purified with TRIzol.
Purified RNA in Smart-seq3 buffer (5% PEG8000, 0.1% Triton X-100, 0.5 U/μL RNase inhibitor, 0.5 μM Smart-seq3 oligo-dT primer, and 0.5 mM dNTP) was incubated at 70°C for 2 min to denature the RNA. Next, reverse transcription mix (25 mM Tris-HCl pH 8.3RT, 30 mM NaCl, 1 mM GTP, 2.5 mM MgCl2, 8 mM DTT, 0.5 U/μL RNase inhibitor, 2 μM TSO and 2 U/μL of maxima H-minus reverse transcriptase enzyme) was added to each sample. Reverse transcription and template switching were carried out at 42 °C for 90 min followed by 10 cycles of 50 °C for 2 min and 42 °C for 2 min. The reaction was terminated by incubating at 85 °C for 5 min. PCR pre-amplification was performed by directly adding KAPA HiFi HotStart ReadyMix and 0.5 μM forward primer and 0.1 μM reverse primer to a 50 μL reaction volume. The PCR was performed with the following cycle: 3 min at 98 °C for initial denaturation, 5–9 cycles (depending on the starting amount of RNA) of 20 s at 98 °C, 30 s at 65 °C, and 4 min at 72 °C. Final elongation was performed for 5 min at 72 °C. After PCR the cDNA was purified twice with 0.6x SPRIselect beads (Beckman Coulter, CA). Next, 600 pg of preamplified cDNA were tagmented with Tn5 transposase (Lucigen) pre-loaded with suitable adapters. Libraries were further amplified for 14 cycles using custom Nextera-compatible primers with different indexes with KAPA HiFi HotStart ReadyMix. Libraries were loaded at 2.4 pM and sequenced on an Illumina Nextseq 500. The read configuration was 38 (read1), 8 (index1), 8 (index2), 38 (read2).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Reads were mapped using STAR 2.7.3a (Dobin et al., 2013) to the Drosophila genome assembly dm6.
Improperly paired reads were removed from bam files using samtools.
Reads per gene annotated in the FlyBase release 2019_05 were computed using an in-house R script based on the GenomicRanges (Lawrence et al., 2013) function summarizeOverlaps, which counts the number of reads overlapping the exons of each gene in the default "union" mode.
Genome_build: dm6
Supplementary_files_format_and_content: read_counts.txt provides the raw read counts for each gene produced using the specified data processing steps.

Submission date

Jul 06, 2021

Last update date

Feb 20, 2023

Contact name

Emily Shields

E-mail(s)

[email protected]

Organization name

University of Pennsylvania

Street address

3400 Civic Center Blvd