GSM6753415: Female, KO, rep 1, run #1; Nothobranchius furzeri; RNA-Seq
Gonads were dissected from each fish according to (Astre, Moses & Harel, 2021) and kept on ice in full medium (L15, 1% penicillin-streptomycin, 50 μg/μl gentamicin, 15% FBS). When all dissections were complete, organs were washed once with L15, and media was replaced with digestion media (400 μl 0.25% trypsin) at 28°C for 2 hours. During the incubation, additional mechanical dissociation was applied by pipetting up and down periodically with a glass Pasteur pipette, followed by addition of 800μl of full medium to stop enzymatic digestion. Dissociated cells were passed through a 100μm cell strainer prior to FACS. For live/dead sorting by FACS, cells were stained with Propidium-iodide (PI, 1 μg/ml), incubated for 5 minutes, and live cells were sorted according to PI intensity using an Aria III Sorter (BD). Live cells were used for single cell RNA sequencing. Microfluidic operations were performed using the Fluigent microfluidic pump system in a microfluidic device, that was fabricated by soft lithography following standard InDrop protocols (Klein et al., 2015; Mazutis et al., 2013), using some adaptations (Bavli et al., 2021; Sun et al., 2021). Cells were loaded at a concertation of 500,000 cells/ml in PBS, with a 10% v/v OptiPrep Density Gradient Medium (Sigma-Aldrich, cat #D1556), and were maintained in suspension using a magnetic micro-stirrer bar placed within the tube. Barcoded beads were prepared as previously described (Bavli et al., 2021; Sun et al., 2021). The reverse transcription/lysis mix consisted of 60 µL 5X First-Strand buffer (SuperScript™ III Reverse Transcriptase Kit, Invitrogen #18080044), 9 µL 10% (v/v) IGEPAL CA-630 (Sigma-Aldrich, Cat #I8896), 6 µL 25 mM dNTPs (NEB, Cat #N0446S), 10 µL 0.1 M DTT (SuperScript™ III Reverse Transcriptase Kit, Invitrogen Cat #18080044), 15 µL 1 M Tris-HCl (pH 8.0) (Sigma-Aldrich, Cat #T2319), 10 µL murine RNase inhibitor (NEB, Cat #M0314), 15 µL SuperScript™ III RT enzyme (200 U/µL, Invitrogen Cat #18080044), and 25 µL nuclease-free water (Sigma-Aldrich, Cat #W4502). The carrier oil was a mixture of HFE-7500 with 2% (w/w) fluorosurfactant (RAN Biotechnologies).   During a microfluidics run, all reagents, including the cell suspension, encapsulated cells, reverse transcription/lysis mix, and collection tubes were kept on ice. For each sample, three different collections were performed separately, to be used as technical replicates. Flow rates were 100 μL/h for both the cell suspension and reverse transcription/lysis mix, 25 μL/h for barcoded acrylamide beads (containing photocleavable barcoding poly-T primers), and 150 μL/h for carrier oil to produce 2 nL drops. Following the completion of the microfluidics stage, the collection tubes were exposed to UV for 10 min (6.5 J/cm2 of a 365-nm), to release photocleavable barcoding primers from the beads. Next, the UV-exposed emulsion was transferred to a reverse transcription reaction (50 °C for 2 h followed by 15 min at 70 °C for inactivation), and was demulsified to release the barcoded cDNA from the droplets by adding perfluoro-1-octanol (50 μl PFO, Sigma-Aldrich, cat #370533).   The upper aqueous phase containing the barcoded cDNA was transferred to a new well, followed by removal of unused primers and primer dimers by adding a 1:1 digestion mix (20 U/µL Exonuclease I, NEB, Cat #M0293; 20 U/µL HinfI enzyme, NEB, Cat #R0155; x1 Exonuclease I Reaction Buffer, NEB, Cat #B0293; x1 CutSmart buffer, NEB, Cat #B7204; and 30 μl of nuclease-free water). Samples were incubated for 1 h at 37 °C and 10 min at 80 °C, purified with AMPure XP beads (Beckman Coulter, Cat #A63882), and eluted in 13.5 μl TE buffer. Second strand synthesis was performed by the NEBNext mRNA Second Strand Synthesis Module (NEB, Cat #E6111) according to the recommended protocols (16 °C for 2.5 h, followed by 20 min at 65 °C). For linear amplification by in vitro transcription, second strand synthesis reaction products were combined with 24 μl T7 High Yield RNA Synthesis Kit (NEB, Cat #E2040) according to the recommended protocols. The reaction was incubated at 37 °C for 13 h, and the resulting RNA was purified with AMPure XP beads, and was used in subsequent library preparation steps.   Next, RNA was fragmented using an RNA fragmentation kit (Invitrogen, Cat #AM8740), followed by purification with AMPure XP beads. The resulting amplified and fragmented RNA was reverse transcribed using a random hexamer primer (PvG748-SBS12-RT (Alajem et al., 2021)). First-Strand synthesis was performed as previously described (Sun et al., 2021), using SuperScript™ III RT enzyme. Following reverse transcription, cDNA was purified with AMPure XP beads, and the resulting libraries were PCR amplified using standard PE1/PE2 full-length primer mix (2p fixed; 2p fixed (+barcode)), containing Illumina library indices for multiplexing, and using 14 amplification cycles (Sun et al., 2021). Amplified libraries were purified (AMPure XP beads), run in 2% agarose gels, and the desired 200-800 bp DNA library fragments were isolated using PureLink™ Quick Gel Extraction Kit (Invitrogen, cat #K210012). Library quality was confirmed by Agilent 2200 TapeStation nucleic acid system (Agilent) using the Agilent High Sensitivity D1000 DS DNA kit. The resulting libraries had an average size of 350-550 bp. Size-selected libraries were diluted to 4 nM, and combined into a pool for paired-end, single index sequencing on the Illumina NextSeq 550 instrument (Illumina 550 High Output v2, 75 cycle kit). Cycle distribution was 45 cycles for Read 1, 35 cycles for Read 2, and 8 cycles for library index read.