GSM6856714: DGRP_Mix_Sleep_Wakefulness_Run5, replicate 1, scRNA-seq; Drosophila melanogaster; RNA-Seq
Brains were dissected in ice-cold Schneider's Medium with 30 mM of the transcription inhibitor Actinomycin D (Sigma-Aldrich 1410). Within 45 min after starting dissections, the brains were dissociated with an enzyme mixture of dispase (3mg/mL), collagenase (100mg/mL) and trypsin-EDTA (0.05%) in a Thermoshaker at 25C for 15 min. The dissociation was reinforced by pipetting the solution at least four times during the incubation. After washing with DPBS 15 mM Actinomycin D, resuspending the cell pellet in 100-200 ml DPBS 0.04% BSA and filtering the cell pellet with a 10mM pluriStrainer ﻿(ImTec Diagnostics_435001050). Library preparations for the single cell RNA-seq was performed using 10X Genomics Chromium Single Cell 3' Kit, v3.1 NextGEM chemistry (10X Genomics, Pleasanton, CA, USA). The cell count and the viability of the samples were assessed using LUNA dual florescence cell counter (Logos Biosystems). For each sample a targeted cell recovery of 10000 cells was aimed for. The dissociated cells from a fly brain are typically quite small (averaging between 1-4 um) and we have seen that this affects the counting accuracy. In order to get consistent counting accuracy, the average of cell counts from three independent measurements were consider for loading the fly cells to the 10X controller. Post cell count and QC, the samples were immediately loaded onto the Chromium Controller. Single cell RNAseq libraries were prepared using manufacturers recommendations (Single cell 3' reagent kits v3.1 user guide; CG000204 Rev D), and at the different check points the library quality was accessed using Qubit (ThermoFisher) and Bioanalyzer (Agilent). For a targeted sequencing saturation of 50-60%, sequencing was performed at a depth of 30,000 - 60,000 reads per cell and single cell libraries were sequenced either on Illumina's NovaSeq 6000 platform or HiSeq 2500 platform using paired-end sequencing workflow and with recommended 10X; v3.1 read parameters (28-8-0-91 cycles). Single-cell RNA-seq (10X Genomics)