title: GSM8880823: First tarsal segment, female, 12h APF, scRNA-seq; Drosophila melanogaster; RNA-Seq
library_construction_protocol: We prepared our single-cell suspensions from dissected first tarsal segment tissue following the protocol we previously developed as part of our cell atlas (Hopkins et al., 2023 PLOS Biology) and which is available on protocols.io (dx.doi.org/10.17504/protocols.io.x54v9dzbmg3e/v2). Briefly, white prepupae were collected, sexed, and transferred to folded kimwipe wet with water (300μl for 12h, 450μl for 20h, and 750μl for 36h) held inside a petri dish. The dish was moved to an incubator at 25°C on a 12:12 cycle and the pupae left to age. Pupae were removed from puparia using forceps and placed on top of a water-soaked kimwipe 1 h before the desired age was reached (e.g., 23 h after collection for the 24 h sample). Pupae were then placed ventral side up on tape, the base of the abdomen pierced to release fluid pressure, and the foreleg removed at the tibia/tarsal joint. The dissected leg was moved to a piece of tape, covered in a drop of 1X Dulbecco's PBS (DPBS, Sigma, D8537), and a Micro Knife (Fine Science Tools, 10318–14) used to sever at the midpoint 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 10μl tip. Once 65-70 segments were 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 2 mg/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. For the 36h APF sample, we incubated for 45 minutes to overcome the greater resistance to dissociation at this point. The dissociation buffer was then removed from the well and replaced with 50μl of room temperature DPBS before the suspension was pipetted up and down 20-40x using a 200μl, widebore, low bind, freshly BSA coated tip (Thermo Fisher Scientific, 2069G) on a 50μl pipette set to 40μl. The solution was then pipetted up and down a further 20-40x using a flame-rounded, BSA-coated 200μl tip, again on a 50μl pipette set to 40μl. Next, the solution was transferred to a 2mL, low-bind, wide-bottomed tube on ice. 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 × 5μl aliquots. 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 manufacturer's instructions.  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. Libraries from the 16h, 20h, 24h, 30h, and 36h APF samples were sequenced on a NovaSeq 6000 sequencer (Illumina, San Diego, CA) with paired-end 150 bp reads. The 12h APF libraries were sequenced in a 28 x 10 x 10 x 90 bp configuration on an AVITI sequencer (Element Biosciences, San Diego, CA).  In each case, the sequencing generated approximately 50,000 reads per cell and 500 million reads per library .