GSM5170787: DNeg_rep2; Drosophila melanogaster; RNA-Seq
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). , Dissociated, live lymph gland cells were sorted using the BD FACSARIA-H. Gates and compensation were based on single color controls. Cells were sorted into 300 µL of DNA/RNA Shield (Zymo) in DNA LoBind tubes and frozen at -80°C prior to RNA extraction. Total RNA was extracted from each sample using the Quick-RNA Microprep Kit (Zymo). RNA quality control was performed using the Agilent 4150 TapeStation system. 3' mRNA capture: cDNA libraries were prepared using KAPA Stranded mRNA-seq kit (KAPA Biosystems)
GEO
GSM5170787