title: GSM4699211: Plate_389_Q8_SS62862 (Single-cell RNA-Seq); Drosophila melanogaster; RNA-Seq
library_construction_protocol: For each sample, 60-100 brains were dissected in freshly prepared, ice cold Adult Hemolymph Solution (AHS, 108 mM NaCl, 5 mM KCl, 2 mM CaCl2, 8.2 mM MgCl2, 4 mM NaHCO3, 1 mM NaH2PO4, 5 mM HEPES, 6 mM Trehalose, 10 mM Sucrose), and the major tracheal branches removed. The brains were transferred to a 1.5 ml Eppendorf tube containing 500 μl 1 mg/ml Liberase DH (Roche, prepared according to the manufacturer's recommendation) in AHS, and digested for 1 h at room temperature. The Liberase solution was removed and the brains washed twice with 800 microliters ice cold AHS. The final wash was removed completely and 400 μl of AHS+2% Fetal Bovine Serum (FBS, Sigma) were added. The brain samples were gently triturated with a series of fire-polished, FBS-coated Pasteur pipettes of descending pore sizes until the tissue was homogenized, after which the tube was allowed to stand for 2-3 m so that the larger debris could settle. For hand sorting, the cell suspension was transferred to a Sylgard-lined Glass Bottom Dish (Willco Wells), leaving the debris at the bottom of the Eppendorf tube, and distributed evenly in a rectangular area in the center of the plate with the pipet tip. The cells were allowed to settle for 10-30 m prior to picking. Fluorescent cells were picked with a mouth aspirator consisting of a 0.8 mm Nalgene Syringe Filter (Thermo), a short stretch of tubing, a plastic needle holder, and a pulled Kwik-Fil Borosilicate Glass capillary (Fisher). Cells picked off the primary plate were transferred to a Sylgard-lined 35 mm Mat Tek Glass Bottom Microwell Dishes (Mat Tek) filled with 170 μl AHS+2%FBS, allowed to settle, and then re-picked. Three washes were performed in this way before the purified cells were picked and transferred into 50 μl buffer XB from the PicoPure RNA Isolation Kit (Life Technologies), lysed for 5 m at 42˚C, and stored at -80˚C. For FACS, the samples were triturated in AHS+2%FBS that was run through a 0.2 μm filter, and the cell suspension was passed through a Falcon 5 ml round-bottom tube fitted with a 35 μm cell strainer cap (Fisher). Samples were sorted on a SONY SH800 cell sorter gated for single cells with a fluorescence intensity exceeding that of a non-fluorescent control. For bulk samples, GFP+tdTom+ events were purity sorted directly into 50 microliters PicoPure XB buffer, the sample lysed for 5 m at 42˚C, and stored at -80˚C. For low-cell samples, replicates of 40 cells were either purity sorted directly or yield sorted to enrich for GFP+tdTom+ cells followed by a purity sort into 3 μL Lysis Buffer (0.2% Triton X-100, 0.1 U/μL RNAsin) and flash frozen on dry ice. For single-cell (scRNA-Seq), individual cells were purity sorted directly into a 96-well plate containing 3 μL Lysis Buffer per well. For bulk samples, total RNA was extracted from 100-500 pooled cells using the PicoPure kit (Life Technologies) according to the manufacturer's recommendation, including the on-column DNAse1 step. The extracted RNA was converted to cDNA and amplified with the Ovation RNA-Seq System V2 (NuGEN), and the yield quantified by NanoDrop (Thermo). The cDNA was fragmented and the sequencing adaptors ligated onto the fragments using the Ovation Rapid Library System (NuGEN). Library quality and concentration was determined with the Kapa Illumina Library Quantification Kit (KK4854, Kapa Biosystems), and the libraries were pooled and sequenced on an Illumina NextSeq 550 with 75 base pair reads. Sequencing adapters were trimmed from the reads with Cutadapt (Martin, 2011) prior to alignment with STAR (Dobin et al., 2013) to the Drosophila r6.17 genome assembly (Flybase). The resulting transcript alignments were passed to RSEM (Li and Dewey, 2011) to generate gene expression counts. For low-cell and single-cell samples, one microliter of harsh lysis buffer (50 mM Tris pH 8.0, 5 mM EDTA pH 8.0, 10 mM DTT, 1% Tween-20, 1% Triton X-100, 0.1 g/L Proteinase K (Roche), 2.5 mM dNTPs (Takara), and ERCC Mix 1 (Thermo-Fisher) diluted to 1e-7) and 1 μl10 μM barcoded RT primer were added, and the samples were incubated for 5 m at 50˚C to lyse the cells, followed by 20 m at 80˚C to inactivate the Proteinase K. Reverse transcription master mix (2 μL 5X RT Buffer (Thermo-Fisher), 2 μL 5M Betaine (Sigma-Aldrich), 0.2 μL 50 μM E5V6NEXT template-switch oligo (Integrated DNA Technologies), 0.1 μL 200 U/μL Maxima H-RT (Thermo-Fisher), 0.1 μL 40U/μL RNAsin (Lucigen), and 0.6 μL nuclease-free water (Thermo-Fisher) was added to the lysis reaction and incubated at 42˚C for 1.5 h, followed by 10 m at 75˚C to inactivate the reverse transcriptase. PCR was performed by adding 10 μL 2X HiFi PCR Mix (Kapa Biosystems) and 0.5 μL 60 μM SINGV6 primer and incubating at 98˚C for 3 m, followed by 20 cycles of 98˚C for 20 s, 64˚C for 15 s, 72˚C for 4 m, and a final extension of 5 m at 72˚C. Groups of 8 reactions were pooled to yield ~250 μL and purified with Ampure XP Beads (0.6x ratio; Beckman Coulter), washed twice with 75% Ethanol, and eluted in 40 μL nuclease-free water. The DNA concentration of each sample was determined using Qubit High-Sensitivity DNA kit (Thermo-Fisher). To prepare the Illumina sequencing library, 600 pg cDNA from each pooled sample was used in a modified Nextera XT library preparation (Illumina) (Soumillon et al., 2014) using the P5NEXTPT5 primer and extending the tagmentation time to 15 m. The resulting libraries were purified according to the Nextera XT protocol (0.6x ratio) and quantified by qPCR using Kapa Library Quantification (Kapa Biosystems). Six to eight sequencing libraries were loaded on a NextSeq High Output flow cell reading 26 bases in Read 1, including the spacer, sample barcode and UMI, 8 bases in the i7 index read, and 50 bases in Read 2 representing the cDNA fragment from the 3' end of the transcript.