title: GSM5170763: dissociated lymph gland blood cells_rep2; Drosophila melanogaster; RNA-Seq
library_construction_protocol: Lymph glands were dissected out of early 3rd instar larvae,  their anterior lobes were removed, and the primary lobes were transferred to a watchglass filled with dissecting buffer on ice. After dissection, the lymph glands were dissociated as previously described with some modifications (Harzer et al. 2013; Khan et al. 2016). After being washed with MDS buffer twice, these tissues were transferred to 1.5 mL DNA LoBind tubes (Eppendorf) and incubated with 200 µL of dissociation solution containing 1 mg/mL of papain (Sigma, P4762) and 1 mg/mL of collagenase (Sigma, C2674) in Schneider's medium. They were dissociated for 15 minutes in a shaking incubator at 25°C, 300 rpm. Next, 500 µL of cold Schneider's medium was added and the suspension was gently pipetted up and down using a low-binding 1000 µL tip (Olympus Plastics) 20 times for mechanical dissociation. After centrifugation at 3000 rpm for 5 minutes, cells were resuspended and washed with 500 µL of 1X PBS (Corning, MT21040CV) containing 0.04% of UltraPure BSA (Invitrogen, AM2616) and then passed through a 35 micrometer cell strainer (Falcon 352235). For preparation of the single cell RNA-Seq sample, the cell suspension was concentrated by centrifuging and resuspending in a lower volume of PBS containing 0.04% BSA (30 µL). Cell concentration and viability were assessed using the Countess II automated cell counter (Applied Biosystems). The samples with final concentration of more than 650 cells/µL and viability of more than 85% were used for single cell RNA-Seq. 3' mRNA capture: Libraries were prepared using the instructions for 10X Genomics Single Cell 3' GEX version 3. 5,000 cells were targeted for each sample.
attributes.tag: GEO Accession
attributes.value: GSM5170763