Jan 16, 2024

Public workspaceSingle cell sequencing preparation in adult Drosophila Brain

DOI
https://dx.doi.org/10.17504/protocols.io.261ged79dv47/v1
  • Mel Feany1,2
  • 1Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Beatrice Weykopf
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DOI: https://dx.doi.org/10.17504/protocols.io.261ged79dv47/v1
Protocol Citation: Mel Feany 2024. Single cell sequencing preparation in adult Drosophila Brain. protocols.io https://dx.doi.org/10.17504/protocols.io.261ged79dv47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 16, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 93630
Keywords: ASAPCRN, preparation in adult drosophila brain, preparation of adult drosophila, adult drosophila brain, adult drosophila, sequencing preparation, single cell, sequencing, cell
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000301
Abstract
This protocol covers the preparation of adult Drosophila Brains for single cell RNAseq
Troubleshooting
Fly brain dissection
15m
Dissect 20 male and 20 female brains from 10-day-old flies on ice-cold Schneider’s medium with FBS (Gibco, filtered 10% FBS)
After a quick spin down, remove the supernatant and wash the brains with ice-cold PBS

Incubate the brain at Temperature25 °C with 300 µl of 0.05% trypsin-EDTA (Fisher Scientific) with continuous pipetting every 5 minutes

Pass solution containing brain chunks through a 25-gauge needle (25G 5/8), without introducing air bubbles, roughly 50 times
After dissociation, the solution was through a 10 µm pluri-select cell strainer (Fisher Scientific), and 400 µl of ice-cold Schneider’s medium containing FBS was added to inactivate trypsin
Centrifuge for Duration00:15:00 at 600xg and remove the  supernatant without disturbing the pellet

15m
Suspend pellet in sterile PBS containing 0.04% BSA
SIngle-cell quantification
15m
Quantify the number of cells and measure viability using an AO-PI reagent (Logos Biosystems)
Prepare sequence libraries
Encapsulate cells, prepare and sequence libraries using the manufacturer's protocol (10X genomics & Illumina Inc.)