GSM5770074: Antenna nuclei Rep2; Aedes aegypti; RNA-Seq
Nuclei extraction from mosquito antennae and max palp were performed as previously described (Li et al. Fly Cell Atlas) with modifications. Prepare fresh homogenization buffer (for recipe, see Li et al. Fly Cell Atlas) and keep on ice. Thaw samples from -80ºC on ice. Spin down samples in 100 µl Schneider's medium using the bench top spinner, discard medium as much as possible. Antennae or Max palp from multiple dissection sessions were combined into a single 1.5 ml EP tube using a low-retention 200 uL filter tip (Rainin 30389240) with ~1 mm from the distal end trimmed using a sterilized and RNAse away-treated (Thermo Fisher 7000TS1) razor blade (VWR 10835-965). And add 100 µl Homogenization butter. Use a pestle motor (Kimble 6HAZ6) to grind the sample for 30s–60s on ice. Add 900 µl homogenization buffer, and transfer 1000 µl homogenized sample into the 1ml dounce (Wheaton 357538). Dounce sets should be autoclaved at 200ºC >5h or overnight. Release nuclei by 20 strokes of loose dounce pestle and 40 of tight dounce pestle. Keep on ice. Avoid bubbles. Filter 1000 µl sample through 5 ml cell strainer (35 µm), and then filter sample using 40 µm Flowmi (BelArt, H13680-0040) into 1.5 ml EP tube. Centrifuge for 10 min at 1000g at 4ºC. Discard the supernatant. Do not disturb the pellet. Re-suspend the nuclei using 500ul 1xPBS/0.5%BSA with RNase inhibitor (9.5 ml 1x PBS, 0.5 ml 10% BSA, 50 µl RNasin Plus). Pipet more than 20 times to completely re-suspend the nuclei. Filter sample using 40 µm Flowmi into a new 5 ml FACS tube and keep the tube on ice. Now the single-nucleus suspensions are ready for FACS. 10x Genomics sequencing libraries were prepared following the standard protocol from 10x Genomics 3' v3.1 kit with following settings. All PCR reactions were performed using the Biorad C1000 Touch Thermal cycler with 96-Deep Well Reaction Module. 13 cycles were used for cDNA amplification and 16 cycles were used for sample index PCR. As per 10x protocol, 1:10 dilutions of amplified cDNA and final libraries were evaluated on Agilent 4200 TapeStation.