A cell atlas of the adult Drosophila midgut

Drosophila guts were dissociated to single cells as previously described (Dutta et al., 2015) with a few modifications. Guts were dissected from 7-day old adult esg-sfGFP/+, pros- Gal4>RFP/+ females. After pulling out the gut, the crop and midgut/hindgut junction (where the Malpighian tubules branch out of the gut) and Malpighian tubules were removed. Five flies at a time were dissected and immediately transferred into cold PBS containing 1% BSA to avoid exposing the midgut tissue to room temperature for a long period of time. Once 40 guts were dissected, they were transferred to a dissection plate and chopped into small pieces using a razor blade. These small fragments were immediately transferred to an eppendorf tube containing 400 ul of 1 mg/ml elastase/PBS solution (Sigma E0258) and incubated on a shaker at 27 ̊C for 30 min. 1% of BSA in PBS (final concentration) was used to stop the digestion reaction and prevent cells from aggregating. The cell suspension was passed through 100 um and then a 40 um cell strainer and loaded on the top of Optiprep with density gradient 1.12 g/ml. Viable cells were isolated from the top layer of the sample after centrifugation at 800xg for 20 min. Cell viability and number were assessed by 0.4% trypan blue and hemocytometer. inDrop and 10x single-cell RNA-sequencing of 10605 cells from the Drosophila midgut