GSM5885225: (Rd): leg, collected Post-molth after amputation, adult animal 56, L side, distal part [Rd_Post_molt_L_56]; Parhyale hawaiensis; RNA-Seq
For the embryonic samples, embryos were dissected in the lid of a 5 ml Protein LoBind Tube (Eppendorf, #0030108302), in 1% BSA in FASW (80 ul). The eggshell was removed with fine forceps (Fine Science Tools, #11254-20), and legs were dissected with borosilicate needles (pulled capillaries; Sutter, #B100-50-15). Samples were transferred in 100 ul of ice-cold lysis solution (Agilent Absolutely RNA Nanoprep kit, #400753), homogenized though brief pipetting, and flash-frozen in liquid nitrogen. RNA extraction was performed with the Agilent Absolutely RNA Nanoprep kit (#400753), following manufacturer's instructions, and eluted in 10 ul of elution buffer. RNA extraction was performed in randomized batches, to avoid shared batch effects in biological replicates. For the adult samples, leg fragments were transferred immediately after dissection in 1.5 ml LowBind tubes with 500 ul of ice-cold lysis buffer (Reliaprep RNA Tissue MiniPrep System - Promega, #Z6111), vortexed, then transferred to a sterile multiwell plate for manual disruption of cuticle with a clean surgical blade. The sample was then re-transferred to the tube, vortexed, and frozen in liquid nitrogen, for then being stored at -80°C until further processing. RNA extraction was randomized, avoiding to process at the same time related samples. RNA was extracted with the Reliaprep RNA Tissue MiniPrep System (Promega, #Z6111), and eluted in 15 ul of nuclease-free water (Invitrogen, #AM9937). 1ng of RNA was used for cDNA synthesis. For samples from embryonic legs, RNA concentration was too low to be directly quantified, 9ul of RNA solution was used for cDNA synthesis over 15 cycles of LD-PCR, using the SMART-Seq v4 Ultra Low Input RNA kit for sequencing (Takara Bio, #634898) and the SeqAmp DNA Polymerase (Takara Bio, #638509); 1 ul of cDNA was then used for Qubit quantification (4.0 HS DNA), measuring in the range of 0.2- 0-7 ng/ul. Libraries were synthesized from 1 ng of cDNA, using the Nextera XT DNA Library Preparation Kit (Illumina, #FC-131-1096; with Dual indexing strategy, i7 and i5), and with a protocol that included an accelerated cooling step on ice after the 55°C step. Quantification and validation of libraries were done with both Qubit 4.0 (HS DNA kit, Thermofisher) and Tapestation D5000 equipment using the D5000 ScreenTape System (Agilent, #5067- 5588 and #5067- 5589).