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Sample GSM6966003

Query DataSets for GSM6966003

Status

Public on Mar 21, 2023

Title

C2_3 neurons, asynA53T, 25d, sample1

Sample type

SRA

Source name

C2_3 neurons

Organism

Drosophila melanogaster

Characteristics

tissue: C2_3 neurons
genotype: C2_3 > asyn[A53T], CD8::GFP

Growth protocol

light-controlled incubator at 25°C and 12-hour light-dark cycle, standard corn meal and molasses food

Extracted molecule

total RNA

Extraction protocol

For 10x library preparation cells were first counted with the FLUNA-FL Dual Fluorescence Cell Counter and viability determined with Acridine Orange/Propidium Iodide dyes (Logos biosystems), which was >93% for all samples, typically 98-100%. We targeted 10 000 captured cells per genotype and sample with Chromium v3.1 chemistry (10x Genomics). For adapted Smart-seq3, 400 GFP+ C2_3 neurons were FAC-sorted for each sample.
For the 5d and 25d 10x datasets, single-cell encapsulation was carried out with the 10x Chromium Next GEM chip; for the 45d 10x dataset with a custom HyDrop chip. Library preparation followed the 10x Single cell 3’ reagent kit v3.1 user guide (CG000204 Rev D). Briefly, reverse transcription was performed on a C1000 Touch Thermal Cycler (Bio Rad) at 53°C 45 min and 85°C 5 min with a 4°C hold, followed by breakage of the single-cell emulsion, Dynabeads MyOne SILAN clean-up and cDNA amplification at 98°C 3 min, 13 cycles of 98°C 3 min - 63°C 20 s - 72°C 1 min, with a final 72°C 1 min extension and 4°C hold. Amplified cDNA was cleaned with SPRIselect (Beckman Coulter). Thereafter cDNA was enzymatically fragmented, followed by end-repair, A-tailing, SPRIselect clean-up, adapter ligation and another SPRIselect clean-up was carried out prior to PCR amplification of the ligation products incorporating unique sample indices at 98°C 45 s, 15 cycles 98°C 20 s - 54°C 30 s - 72°C 20 s - with a final 72°C 1 min extension and 4°C hold. SPRIselect clean-up of the amplified libraries was carried out. At different check points the quality of cDNA and final sequencing library was evaluated using Qubit (ThermoFisher) and Bioanalyzer (Agilent). Libraries were first sequenced shallow to determine library complexity and subsequently sequenced deeper to achieve ~80% sequencing saturation. For the FAC-sorted bulk RNAseq of C2_3 neurons we followed the Smart-seq3 protocol with increased lysis buffer to 7.5 µl including the sorted cell volume, the RT mix volume to 2.5 µl and the final cDNA amplified product to 25 µl in total. Tn5 concentration increased to the Smart-seq2 protocol.
10x Genomics v3.1 scRNAseq and adapted Smart-seq3 for bulk RNAseq

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 2000

Description

adapted Smart-seq3
C2_3_FACS_25d_counts.tsv
C2_3_25d_asynA53T_sample1

Data processing

Pooled sequencing data from 10x libraries was demultiplexed and index-hopping-filter v1.0.1 (10x Genomics) applied. Feature-barcode matrices were computed with Cell Ranger (v4.0.0, 10x Genomics). The fraction of contaminating ambient RNA was automatically determined and corrected with soupX (v1.5.2). Subsequently, individual ambient RNA corrected matrices were loaded into scanpy (v1.8.1) and concatenated into one dataset. Quality control steps included filtering of cells from 5 day old animals at a minimum of 800, 25/45 day old animals 600 detected genes as well as a maximum of 20 000 counts and 15% mitochondrial content.
Reads from the adapted Smart-seq3 libraries were preprocessed with fastp (v0.20.0) followed by alignment and counting with STARsolo (STAR v2.7.9).
Assembly: 4th 2020 FlyBase Drosophila melanogaster release (r6.35)
Supplementary files format and content: tab-delim text and matrix files

Submission date

Jan 24, 2023

Last update date

Mar 21, 2023

Contact name

Patrik Verstreken

E-mail(s)

patrik.verstreken@kuleuven.be

Organization name

VIB-KU Leuven

Street address

Herestraat 49