Illumina HiSeq 2500 sequencing; RNA-seq of Hydra body segments and regenerated Hydra animals
RNA-seq of Hydra body segments and regenerated Hydra animals
Animals of the 105 strain were first bisected at the midpoint between the head and the budding zone. The halves created this way were bisected again in the middle between the head and the previous cut, or the previous cut and the budding zone, respectively, thus creating “body 2” and “body3” segments. Head with tentacles and budding zone with the foot were then removed from the remaining pieces to generate “body1” and “body4” segments. Finally, the budding zone was separated from the foot, using the change of endoderm coloration as a guideline for sectioning. Tentacles were also separated from the head, trying to remove as much of the tentacle tissue as possible. Regenerated animals were left to close in DM for 2hrs then transferred to HM and collected after 72h. The individual segments were lysed immediately after being cut in 350 ul of RL buffer (Single Cell RNA Purification Kit, Norgen), supplemented with 1 % β-mercaptoethanol, frozen on dry ice and stored at -80 ºC for later RNA isolation. Tentacles of a single animal were pooled as one sample. RNA extraction was performed according to the manufacturer's instructions.  cDNA amplification was performed using the SmartSeq2 approach as per the original protocol. Full length cDNA was processed for Illumina sequencing using Tagmentation with an in-house purified Tn5 transposase: 1ng of amplified cDNA was tagmented in TAPS-DMF buffer (10mM TAPS pH8.5, 5mM MgCl2, 10% DMF), at 55C for 7min. Tn5 was then stripped using SDS (0.04% final) and tagmented DNA was amplified using Phusion High-Fidelity DNA Polymerase (ThermoFisher). PCR was performed in the Phusion HF buffer, with a first extension at 72 °C for 3min, followed by 10 cycles of amplification (95 °C – 30 s, 55 °C –30 s, 72 °C – 30s). Commercial Nextera XT indexes were used for the PCR amplification (1/5 dilution).