title: GSM4933139: 48hrAPF_Male; Drosophila melanogaster; RNA-Seq
library_construction_protocol: The tissue was dissected in cold Schneider 2 Drosophila culture medium (S2 medium, Gibco) and transferred to a LoBind tube (Eppendorf), containing 200μl of S2 medium. The tissue was centrifuged at 500g for 5 minutes, and then was washed with 300μl of EBSS (Earle's Balanced Salt Solution), and centrifuged again at 500g for 5 minutes. After centrifugation, the supernatant was replaced with 100μL of papain for disassociation (50 units/ml, Worthington) diluted in EBSS. Brains were dissociated at 25°C in a LoBind tube for 30 min., with pipette mixing to reinforce dissociation every 3 min with a P200 tip during the first 15 minutes, and a P10 tip for the final 15 min. Cells were washed twice with 700μl cold S2 medium containing 10% FBS (Gibco) and centrifuged at 700g for 10 minutes to quench the papain. Cell suspensions were passed through a 30μM pre-separation filter (MiltenyiBiotech). Cell viability and concentration were assessed by hemocytometer using Trypan blue. Single-cell libraries were generated using Single Cell 3' Library & Gel Bead Kit v2, Chip Kit, and the GemCode 10X Chromium instrument (10X Genomics, CA), according to the manufacturer's protocol (Zheng et al. 2017). In brief, single cells were suspended in S2 medium with 10% FBS and the maximum volume of cells, 34μl, was added to a single chip channel. After the generation of nanoliter-scale Gel bead-in-EMulsions (GEMs), the mRNA in GEMs underwent reverse transcription. Next, GEMs were broken, and the single-stranded cDNA was isolated, cleaned with Cleanup Mix containing DynaBeads MyOne Silane beads (Thermo Fisher Scientific). cDNA was then amplified with the following PCR machine settings: 98°C for 3 min, 9 cycles of (98°C for 15s, 67°C for 20s), 72°C for 1 min, held at 4°C. Subsequently, the amplified cDNA was cleaned up with SPRIslect Reagent kit (Beckman Coulter), fragmented, end-repaired, A-tailed, adaptor ligated, and cleaned with SPRIselect magnetic beads between steps. This product was PCR amplified with the following PCR machine settings: 98°C for 45s, 12 cycles of (98°C for 20s, 54°C for 30s, 72°C for 20s), 72°C for 1 min, and hold at 4°C. The library was cleaned and size-selected with SPRIselect beads, followed by Pippin size selection for a 350-450bp library size range. Single cell libraries were sequenced on the Illumina NovaSeq with 150bp paired-end reads on an S2 flowcell. This produced 1,870,220,065 reads.
attributes.tag: GEO Accession
attributes.value: GSM4933139