title: GSM7792315: FW_gill_sample5,replicate1,scRNA-seq; Protopterus annectens; RNA-Seq
library_construction_protocol: The “AE_” samples were collected from terrestrialized lungfish for 33 days. Fresh collected tissues disaggregation and enzymatic dissociation were performed independently. Following dissociation, the resulting suspension was passed through a 40 μm filter (BD # 352340), and the filter was washed with 2 ml cold PBS to ensure efficient digestion and sufficient collection. The filter suspension was transferred to a 15 ml centrifuge tube and centrifuge at 4 ℃ with 300x g for 5 minutes. Then the supernatant was removed, and the centrifugation sediment was washed with 5 ml PBS+0.04%BSA and centrifuged again (4 ℃ at 300x g for 5 minutes), and the centrifugation sediment was rewashed with 5 ml PBS+0.04%BSA. After the centrifugation, the cell pellets were re-suspended in 300 μl PBS+0.04%BSA. All samples were stained, and cell viability was measured using Trypan Blue (Invitrogen) and manually counted using a hemocytometer (INCYTO). The cell suspension concentration was reconstituted to 1000 cells per µL in DPBS for DNBelab C4 system library preparation. Except for two freshwater gill samples, all single-cell RNA-seq libraries were prepared using a DNBelab C Series Single Cell Library Prep Set (MGI, 1000021082) as previously described 122. These included 21 libraries from the lung (eleven libraries of the freshwater group and ten libraries of terrestrialized group) and 27 libraries from the gill (seventeen libraries of the freshwater group and ten libraries of terrestrialized group, libraries) were obtained. Briefly, single-cell suspensions were used for droplet generation, emulsion breakage, bead collection, reverse transcription, and cDNA amplification to generate barcoded libraries. Indexed libraries were constructed according to the manufacturer's protocol. Libraries were constructed and the DNA nanoballs (DNBs) were loaded into the patterned nanoarrays and sequenced on a DNBSEQ-T1 sequencer at the China National GeneBank (Shenzhen, China) with the following sequencing strategy: 41-bp read length for read 1 and 100-bp read length for read 2.Two freshwater gill libraries (FW-gill9 and FW-gill10) were generated with a 10x Genomics Chromium 3′Reagent Kit instructions (v2) and sequenced on a MGISEQ 2000 sequencer. Gill cells were resuspended in PBS with 0.04% BSA and loaded into a 10x Genomics Chromium cell controller to generate a library for sequencing (28 base pairs for read 1 and 98 for read 2).