GSM6620796: First tarsal segment, male, 24h APF, scRNA-seq; Drosophila melanogaster; RNA-Seq
White P1 prepupae (0-1 hour After Puparium Formation, APF) from the DGRP line RAL-517 (Mackay et al., 2012) were collected and sexed based on the presence of testes. Males were transferred to a folded kimwipe, wet with 500µl of water and held inside of a petri dish in an incubator maintained at 25°C on a 12:12 cycle. 1h before the desired age was reached (e.g. 23h after collection for the 24h sample), pupae were removed from puparia using forceps and placed on top of a water-soaked kimwipe. When the desired age was reached, pupae were placed ventral side up on tape. The base of the abdomen was pierced to release some of the fluid pressure and the foreleg removed at the tibia/tarsal joint. The dissected leg was then placed on tape, covered in a drop of 1X Dulbecco's PBS (DPBS, Sigma, D8537), and a scalpel was used to sever at approximately the mid-point of the second tarsal segment. The first tarsal segment was then eased out of the pupal cuticle and transferred to a glass well on ice containing 100µl of 1X DPBS using a BSA-coated tip. We separately prepared two single cell suspensions, each composed of 67 first tarsal segments collected following the procedures described above: one from 24h APF male pupae, the other from 30h APF male pupae. For a given timepoint, once 67 first tarsal segments had been collected, the DPBS was removed from the well and replaced with 100µl of dissociation buffer, which consisted of 10X TrypLE (Thermo Fisher Scientific, A12177-01) with a final concentration of 2µl/mL of collagenase (Sigma, C0130). The well was sealed and submerged in a metal bead bath in an incubator at 37°C for 35 minutes. The dissociation buffer was then removed from the well and replaced with 50µl of room temperature DPBS before the tissues were subjected to mechanical dissociation. For this, the solution was pipetted up and down 20 times using a 200µl, widebore, low bind, freshly BSA coated tip on a 50µl pipette set to 40µl. The tip was fully submerged to avoid bringing air into the suspension. The solution was then pipetted up and down a further 20x using a flame-rounded, BSA-coated 200µl tip, again on a 50µl pipette set to 40µl. Next, the solution was pipetted up and down slowly 3 times using the same flame-rounded tip before 40µl was taken up and transferred to a 2mL, low-bind, wide-bottomed tube on ice. 20µl of DPBS was then slowly dripped around the edges of the well using a different tip to flush the cells into the center. This was then pipetted up and down 3 times using the original flame-rounded 200µl tip and added to the tube. Cell concentration and viability was assayed using an acridine orange/propidium iodide stain and measured using a LUNA-FL Fluorescent Cell Counter averaged across 2 x 5µl aliquots (24h APF sample: 1990 cells/µl, 98% viability; 30h APF sample: 967 cells/µl, 98% viability). Barcoded 3' single cell libraries were prepared from single cell suspensions using the Chromium Next GEM Single Cell 3' kit v3.1 (10X Genomics, Pleasanton, California) for sequencing according to the recommendations of the manufacturer.  The cDNA and library fragment size distribution were verified on a Bioanalyzer 2100 (Agilent, Santa Clara, CA). The libraries were quantified by fluorometry on a Qubit instrument (LifeTechnologies, Carlsbad, CA) and by qPCR with a Kapa Library Quant kit (Kapa Biosystems-Roche) prior to sequencing. The libraries were sequenced on a NovaSeq 6000 sequencer (Illumina, San Diego, CA) with paired-end 150 bp reads.