Single cell RNA sequencing on posterior intestinal tracts and surrounding tissues dissected from living embryos in 4 time-windows from 3.5 hours to 10 hours post-laying
Single cell RNA sequencing on posterior intestinal tracts and surrounding tissues dissected from living embryos in 4 time-windows from 3.5 hours to 10 hours post-laying
Living embryos were collected within 2 hour time windows (3.5-5.5 hours (T1a) 5-7  hours (T1b) 6.5-8.5 (T2) 8-10 hours (T3)). They were dechorionated and gently stuck down on a thin strip of double-sided sticky tape, and the tape mounted on poly-L-lysine coated coverslips. They were covered in cold 0.01% PBS-BSA and dissected by mouth pipetting using sharpened pulled capillary needles as described in 'Broadie, K., Skaer, H. and Bate, M. Whole-embryo culture of Drosophila:development of embryonic tissues in vitro. Rouxs Arch Dev Biol, 1992'. Immediately after dissecting 10 embryos within a 30 mins period, the tissue was pooled and dissociated into single cells by mouth pipetting using pulled glass capillaries while the samples are incubating in the mild cell dissociation buffer TrypLE Express. After 10 mins, cells were fixed in pre-chilled to -20C Methonol to a final concentration of 80:20 Methanol:PBS and stored at -80C. Prior to library preparation, 15 samples of 10 embryos were pooled per collection window. Cells were rehydrated in 0.01% PBS-BSA with the RNAase inhibitor RiboLock and filtered through 20um cell strainer before resuspending, ready for scRNA library preparation. They were covered in cold PBS and dissected by mouth pipetting using sharpened pulled capillary needles as described in  Broadie, K., Skaer, H. and Bate, M. Whole-embryo culture of Drosophila:development of embryonic tissues in vitro. Rouxs Arch Dev Biol, 1992.  After dissecting 10 embryos, the tissue was pooled and dissociated into single cells by incubating in mild TrypLE Express for 10 mins and then mouth pipetting using pulled glass capillaries. Single cell RNA libraries were generated from each of the four single cell suspensions  using the 10x Genomics Chromium single cell 3'reagents kit v3.  Cells were mixed with reagents for GEM formation and loaded onto a Chromium Next GEM Chip B for GEM creation. 100ul of each sample was recovered from the chip and placed into tubes and incubated GEM RT reaction for 45 minutes at 53 degrees and 5 minutes at 85 degrees. Recovery reagent was added to each sample and the aqueous phase recovered. This was then cleaned with magnetic Dynabeads MyOne silane beads by incubation with the beads at room temperature for 10 minutes, 80% ethanol washing of captured beads and finally eluting the sample from the beads. cDNA primers and amplification mix was added and 11 cycles of RT amplification performed. The product was purified with SPRIselect reagent and the recovered sample checked for concentration and profile on an Agilent Bioanalyser high sensitivity chip.  25% of this product was used for library generation. Reagents for fragmentation/end repair were added and the samples heated at 32 degrees for 5 minutes followed by incubation at 65 degrees for 30 minutes. The samples were purified using SPRIselect using a double size selection approach. Adaptor oligos were added by ligation and after a further clean up step, sample indexes (chromium i7 plate single index) were added via 11/12 cycles of PCR, with 12 cycles for samples that had a lower cell count. The final product was cleaned with SPRIselect reagent using a double size selection approach. Single cell RNA libraries were generated from each of the four single cell suspensions  using the 10x Genomics Chromium single cell 3'reagents kit v3.  Cells were mixed with reagents for GEM formation and loaded onto a Chromium Next GEM Chip B for GEM creation. 100ul of each sample was recovered from the chip and placed into tubes and incubated GEM RT reaction for 45 minutes at 53 degrees and 5 minutes at 85 degrees. Recovery reagent was added to each sample and the aqueous phase recovered. This was then cleaned with magnetic Dynabeads MyOne silane beads by incubation with the beads at room temperature for 10 minutes, 80% ethanol washing of captured beads and finally eluting the sample from the beads. cDNA primers and amplification mix was added and 11 cycles of RT amplification performed. The product was purified with SPRIselect reagent and the recovered sample checked for concentration and profile on an Agilent Bioanalyser high sensitivity chip.  25% of this product was used for library generation. Reagents for fragmentation/end repair were added and the samples heated at 32 degrees for 5 minutes followed by incubation at 65 degrees for 30 minutes. The samples were purified using SPRIselect using a double size selection approach. Adaptor oligos were added by ligation and after a further clean up step, sample indexes (chromium i7 plate single index) were added via 11/12 cycles of PCR, with 12 cycles for samples that had a lower cell count. The final product was cleaned with SPRIselect reagent using a double size selection approach.