GSM6267526: Leg, 6months, regenerated; Parhyale hawaiensis; RNA-Seq
For each snRNAseq dataset we collected a pool of 12-16 thoracic T4 and T5 legs from large adult males. The smallest specimens had a body length of ~9 mm (estimated age >12 months) and the average had a body length of ~11 mm (estimated age >18 months). The legs were amputated at the proximal end of the basis (excluding the gills and the coxal plates) using a microsurgical knife (Electron Microscopy Sciences, #72047-30) under anaesthesia in 0.02% clove oil. To compare regenerated and intact legs, T4 and T5 thoracic legs from one side of the animal were ampu- tated and allowed to regenerate and to grow until ~2 months (8 weeks) after amputation in one pool of individuals, or ~6 months (24 weeks) after amputa- tion in a separate pool. The contralateral legs were left intact and used as controls. We then collected the sets of regenerated and control T4 and T5 legs by cutting them off at the basis, and dissected them (cut the podomeres lengthwise) in a chilled hypotonic buffer containing 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris buffer pH 8.0 and 1 μM DTT and supplemented with 1x protease inhibitor (Sigma, #11873580001), 0.4 U/μl RNasein (Invitrogen, #AM2682), 0.2 U/μl SUPERase-In RNase inhibitor (Invitrogen, #AM2694), 1 mM spermine and 0.1 M spermidine (modified from (Krishnaswami et al., 2016)). The tissue was extruded from the cuticle by gently scraping and peeling using a microsurgical blade and fine forceps (Fine Science Tools, #10316-14 and #11251-20) under a dissecting stereoscope. Triton X-100 was then added to a final concentration of 0.3 % and the tissues were mechan- ically disrupted by gently pipetting 25 times through a 200 μl filter tip with a cut tip. These steps were carried out in 300 μl of buffer, in the lid of a 5 ml DNA LoBind tube (Eppendorf, #0030108302) kept cold on the surface of a frozen metal plate. The disrupted tissue samples were then transferred to a 1.5 ml DNA LoBind Eppendorf tube (Ependorf, #0030108051), and large pieces of cuticle were let to settle down. The supernatant containing dissociated nuclei was transferred to a new LoBind tube, in which 600 μl fresh hypotonic buffer were added. The nuclei were collected by centrifugation at 500 rcf at 4 ̊C for 6 minutes on a 5424 R Eppendorf microcentrifuge. Centrifugation was kept to a minimum, which result- ed in a loose pellet, because longer or faster centrifu- gations result in lower quality nuclei. The supernatant was discarded and the pellet was washed with 600 μ l 1x Dulbecco's phosphate-buffered saline (DPBS) supplemented with 2% bovine serum albumin (BSA). After a second centrifugation, all the supernatant except 25 μL was removed and an equal volume of 3x DPBS with 2% BSA was added to reach a final concentration of 2x DPBS (we observed that nuclei are better preserved but they are more difficult to pellet in 2x DPBS). To remove aggregates, the sam- ples were filtered through 10 μm cell strainer (PluriSelect Mini strainer) to generate single-nucleus suspensions. All the steps were performed in the cold and samples were kept on ice until nucleus capture. The quality of the nuclei was assessed by staining with trypan blue and nuclei counts were made using a Malassez haemocytometer. Single nucleus RNA sequencing was performed using the 10x Genomics single-cell capturing system. Nuclei suspensions, including 20,000 to 25,000 nuclei, were loaded on the 10x Genomics Chromium Controller following the manu- facturer's instructions, within 1 hour of preparation. Single nucleus cDNA libraries were prepared using the Chromium Single Cell 3' Reagent Kit according to the manufacturer's instructions (chemistries v3 and v3.1). Libraries were sequenced on an Illumina NextSeq500 (28 bp for R1 and 122 bp for R2) and 248 to 315 million reads were obtained per library.