title: GSM9561009: Ensheathing Glia, Pink1 KO, Batch 2, Replicate 1; Drosophila melanogaster; RNA-Seq
library_construction_protocol: Ten brains of flies 5 ± 1 days old were collected, with most of the laminae removed. Dissections were performed in ice-cold PBS that contained 5 μM Actinomycin D (Sigma) to inhibit changes in gene expression during tissue processing. For the dissociation of brain tissue, a mix of Dispase I (0.6 mg/ml) (Sigma), Collagenase I (30 mg/ml) (Thermo Fisher), Trypsin (0.5x) (Thermo Fisher), and 5 μM Actinomycin D was used. Immediately after dissociation, cells were sorted using a FACSAria Fusion (BD Biosciences) flow cytometer with a 100 µm nozzle at 20 psi. Live (DAPI-negative), GFP-positive cells were sorted into 96-well PCR plates at 300 cells per well (3 wells per genotype) using a 4-way purity mask. 3 ul of cell lysis buffer (0.1% Triton X-100, Sigma Aldrich; 1U/ul of RNase Inhibitor, RNase OUT, Thermo Fisher; 2.5 uM Oligo dT(25), IDT; and 2.5 mM dNTP, Promega) was aliquoted in triplicate to 96 well plates (4titude, Cat. No. 4TI-0960/C). Roughly 300 cells were sorted into the wells with lysis buffer and the plate was spun down at 2000xg for 1 min prior to storage at - 80*C. For the first strand synthesis, the Smart-Seq 2 plates were thawed to room temperature for a minute and subsequently spun down at 2000 xg for minute. The RNA denaturation is carried out at 72*C for 10 mins and then flash cooled on ice for 5 mins. First strand synthesis mix (1X SuperScript II first strand synthesis buffer, Thermo Fisher; 5 mM DTT, Thermo Fisher; 100 U SuperScript II reverse transcriptase enzyme, Thermo Fisher; 10 U RNAse OUT, Thermo Fisher; 1M Betaine, Sigma Aldrich; 6 mM MgCl2, Thermo Fisher; and 1 uM TSO, IDT Technologies) is added to the cell lysis mix to a total volume of 10 ul. The First strand synthesis reaction was carried out with following program: 42*C for 90 mins; 10 cycles of {50*C for 2 mins; 42*C for 2 mins}; 72*C for 15 mins; 4*C indefinite hold. PCR amplification of the first strand product was performed by adding 15 ul of the PCR mix to the first strand product (1X KAPA HiFi HotStart Ready Mix, Roche; and 0.1 uM of IS PCR primer, IDT Technologies). 22 cycles of the following PCR program was used to amplify the first strand product: 98*C for 3 mins; 22 cycles of {98*C for 20 secs; 67*C for 15 secs; 72*C for 6 mins}; 72*C for 5 mins; 4*C indefinite hold. 20 ul of Ampure XP was added to each well and mixed. Standard Ampure XP purification was carried out as per manufacturer's recommendation and the cDNA library was eluted in 17.5 ul of Elution buffer. 17 ul of the eluted library was transfered to fresh 96 well plate (4titude, Cat. No. 4TI-0960/C). 5 ul of Tn5 tagmentation mix (1X Tagment DNA buffer, Illumina; ATM mix, Illumina and cDNA library 1 ng) was prepared for cDNA fragmentation. Tn5 tagmentation was performed with following program: 55*C for 10 mins; 4*C indefinite hold. Tagementation reaction was stopped by quenching the reaction with 1.25ul of NT buffer for 5 mins (Illumina). 1.25 ul of the i5 and i7 Illumina indexes were added to the plates. Finally 3.75 ul of NPM master mix was added to the plate and mixed well and put for the index PCR amplification: 72*C for 3 mins; 95*C for 30 secs; 12 cycles of {95*C for 10 secs; 55*C for 30 secs; 72*C for 30 secs}; 72*C for 5 mins, and 4*C indefinite hold. The indexed PCR products were pooled in a single tube, and 0.8X Ampure XP purification (Beckman Coulter) was carried out as per the manufacturer's recommendation, and finally, the sequencing library was eluted in 35.5 μl of Elution buffer (Qiagen). SMART seq 2 libraries were sequenced on the NextSeq 500 (Illumina) sequencing platform. Sequencing was done as per the protocol recommendations: paired-end read of 76 bps (read 1), 76 bps (read 2), 8 bps (index 1) and 8 bps (index 2). For a targeted sequencing depth of ~30 million reads per sample.