GSM5343857: HYR__81e131__20210225_R74_S2; Drosophila melanogaster; RNA-Seq
50 adult brains were dissected in DPBS and transferred to a tube containing 100 μl of cold DPBS solution. Samples were centrifuged at 500 rcf for 1 min and the supernatant was replaced by 50 μL of dispase (3 mg/mL, Sigma-Aldrich, D4818, 2mg) and 75 μl of collagenase I (100 mg/mL, Invitrogen, 17100-017). Brains were dissociated in a Thermoshaker (Grant Bio PCMT) at 500 rpm for 2 h at 25°C, with pipette mixing every 15 min. Cells were subsequently washed with 1000 μl of cold DPBS solution and centrifuged at 500 rcf for 5 min at 4°C and resuspended in 250 μl of DPBS with 0.04% BSA. Cell suspensions were passed through a 10 μM pluriStrainer (ImTec Diagnostics, 435001050). Cells were sorted based on viability and mCherry positivity using the Sony MA900 cell sorter. Sorted cells were collected into Eppendorf tubes pre-coated with 1% BSA. Cells were resuspended in 85 μL of RT mix (1x Maxima RT Buffer, 0.9 mM dNTPs, 25 mM DTT, 1.3 mM GTP, 15 % Optiprep, 1.3 U/uL RNAse inhibitor, 15 U/uL Maxima hRT, 12.5 uM TSO, 4.4% PEG-8000). RT mix was co-encapsulated with 35 μL of freshly thawed HyDrop-RNA beads in RAN oil on the Onyx microfluidics platform. The resulting emulsion was collected in aliquots of 50 μL total volume and thermocycled according to the RT program (42 °C for 90 min., 11 cycles of [50 °C for 2 min., 42 °C for 2 min.], 85 °C for 5 min., followed by a final hold on 4 °C). 125 μL of recovery agent (20% PFO in HFE), 55 μL of GITC Buffer (5 M GITC, 25 mM EDTA, 50 mM Tris-HCl pH 7.4) and 5 μL of 1 M DTT was added to each separate aliquot of 50 μL thermocycled emulsion and incubated on ice for 5 minutes. 99 μL of Ampure XP beads was added to the aqueous phase and incubated for 10 minutes. Ampure beads were pelleted on a Nd magnet and washed twice with 80% EtOH. Elution was performed in 50 μL of EB-DTT-Tween (10 mM DTT, 0.1% Tween-20 in EB). Exonuclease treatment was performed by adding 3 μL of 10x NEBuffer 3.1, 3 μL of ExoI, and 4 μL of dH2O to 30 μL of eluted library. The ExoI reaction mix was incubated at 37 °C for 5 min., 80 °C for 1 min. followed by a final hold at 4 °C. 2 μL of 1 M DTT was added and a 0.8X Ampure XP purification was performed according to manufacturer's recommendations. cDNA was eluted in 40.5 μL of EB-DTT (10 mM DTT in EB) and added to ISPCR mix (40 μL library, 50 μL 2x KAPA HiFi, 10 μL 10 uM TSO-P primer). PCR cycling was performed according to the ISPCR program (95 °C for 3 min., 13 cycles of [98 °C for 20s, 63 °C for 20s, 72 °C for 3 min.], 72 °C for 5 min. followed by a final hold at 4 °C. 2 μL of 1M DTT was added and a 0.6x Ampure XP purification was performed according to manufacturer's recommendations. cDNA was eluted in 28.5 μL of EB-DTT. Final sequencing library was prepared according to the following customised NEB Ultra II FS protocol. 80 ng of amplified cDNA was fragmented in Ultra II fragmentation mix (26 μL of amplified cDNA, 7 μL of NEBNext Ultra II FS Reaction Buffer, 2 μL of NEBNext Ultra II FS Enzyme Mix) on the following thermocycling program: 37 °C for 10 min., 65 °C for 30 min. and a final hold at 4 °C. 15 μL of EB was added and a 0.8X Ampure purification was performed according to manufacturer's recommendation and eluted in 35 μL. Fragmented library was adapter-ligated in NEBNext Ultra II adapter ligation mix (35 μL of fragmented library, 30 μL of NEBNext Ultra II Ligation Master Mix, 1 μL of NEBNext Ligation Enhancer, 2.5 μL of NEBNext Adapter for Illumina) at 20 °C for 15 min., with 4 °C final hold. 28.5 μL of EB was added and a 0.8X Ampure purification was performed according to manufacturer's recommendation and eluted in 30 μL. Eluted library was amplified in PCR master mix (50 μL 2x KAPA HiFi, 10 μL 10 uM Hy-i7 primer, 10 μL 10 uM Hy-i5 primer, 30 μL eluted library) in the following thermocycling program: 95 °C for 3 min., 13 cycles of [98 °C for 20 s, 64 °C for 30 s, 72 °C for 30 s], 72 °C for 5 min. and a final hold at 4 °C. Sequencing-ready library was purified using a 0.8X Ampure purification and eluted in 30 μL of EB.
GEO
GSM5343857