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Sample GSM8172231

Query DataSets for GSM8172231

Status

Public on Mar 31, 2024

Title

Drosophila melanogaster epidermal cells 1 [epi-1]

Sample type

SRA

Source name

epidermal cells

Organism

Drosophila melanogaster

Characteristics

cell type: epidermal cells
genotype: WT

Treatment protocol

Epidermal cells were isolated from control (GMR38F11-GAL4, UAS-2xEGFP/+) or miR-14 mutant (miR-14-delta1/miR-14-delta1; GMR38F11-GAL4, UAS-2xEGFP/+) larvae

Growth protocol

Larvae were reared at 25 C under 12 h light / dark cycle on standard cornmeal/molassess media and isolated for dissection at 96 h after egg laying.

Extracted molecule

polyA RNA

Extraction protocol

Larvae with cytoplasmic GFP expressed in different epidermal subsets were microdissected and dissociated in collagenase type I (Fisher 17-100-017) into single cell suspensions, largely as previously described, with the addition of 1% BSA to the dissociation mix. After dissociation, cells were transferred to a new 35 mm petri dish with 1 mL 50% Schneider’s media, 50% PBS supplemented with 1% BSA. Under a fluorescent stereoscope, individual fluorescent cells were manually aspirated with a glass pipette into PBS with 0.5% BSA, and then serially transferred until isolated without any additional cellular debris present. Ten cells per sample were aspirated together, transferred to a mini-well containing 3ul lysis solution (0.2 % Triton X-100 in water with 2 U / µL RNAse Inhibitor), lysed by pipetting up and down several times, transferred to a microtube, and stored at -80º C. For the picked cells, 2.3 µL of lysis solution was used as input for library preparation.
RNA-Seq libraries were prepared from the picked cells following the Smart-Seq2 protocol for full length transcriptomes. To minimize batch effects, primers, enzymes, and buffers were all used from the same lots for all libraries. Libraries were multiplexed, pooled, and purified using AMPure XP beads, quality was checked on an Agilent TapeStation, and libraries were sequenced as 51-bp single end reads on a HiSeq4000 at the UCSF Center for Advanced Technology.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

epi-1

Data processing

Reads were demultiplexed with CASAVA (Illumina) and read quality was assessed using FastQC and MultiQC. Reads containing adapters were removed using Cutadapt version 2.4 and reads were mapped to the D. melanogaster transcriptome, FlyBase genome release 6.29, using Kallisto version 0.46.0 with default parameters.
Assembly: FlyBase genome release 6.29
Supplementary files format and content: FASTQ

Submission date

Mar 27, 2024

Last update date

Mar 31, 2024

Contact name

Jay Z Parrish

E-mail(s)

jzp2@uw.edu

Phone

2066851203

Organization name

University of Washington