title: GSM7770353: RasV12 UAS-RasV12(4847/Bl) , ScRNA-seq; Drosophila melanogaster; RNA-Seq
library_name: GSM7770353
library_construction_protocol: Ten pairs of salivary glands per genotype were dissected into individual droplets of 200 µl PBS. A fluorescent microscope Leica DMi8 (Leica, Germany) was used to locate the attached hemocytes at the surface of the salivary gland. The hemocytes were collected using micromanipulator TransferMan NK2 (Eppendorf, Germany) with ø 7 µm collection capillary and separated by piezo-vibrator PiezoXpert (Eppendorf, Germany). Single-cell state hemocytes were individually transferred to 2.3 µl of lysis buffer (38) and each sample library was prepared cDNA libraries of single attached hemocytes were generated using a modified version of the Smart-seq2 protocol,  cDNA synthesis was performed using universal primers, and PCR amplification was carried out over 24 cycles. cDNA products were subsequently purified using CA beads Combinatorial indexing via tagmentation was carried out in 96-well plates using 200 pg (measured in a Qubit fluorometer) of amplified cDNA, for a final volume of 10 mL/well. cDNA fragmentation using Tn5 transposase was carried out for 20 min on ice using the Illumina Nextra XT DNA sample preparation kit. Ligation and amplification of adaptors were carried out over 15 cycles in a final volume of 25 mL/well, Tagmented and barcoded amplicons were then purified using CA beads for size selection. Quality control and fragment size distribution of the cDNA libraries were performed on a Bioanalyzer with the Agilent high-sensitivity DNA chip (catalog no. 5067-4626). Concentrations of each sample of cDNA libraries were measured on a PicoGreen 96-well plate NucleoScan fluorometer using a high-sensitivity double-stranded DNA (dsDNA). To perform library dilutions, the average fragment sizes of all cDNA libraries were measured for a final concentration of 2 nM in each sample. Finally, cDNA libraries were pooled and sequenced using Illumina NextSeq with 75-bp paired-end reads