title: GSM5740462: 105-G-Tb-4; Hydra vulgaris; RNA-Seq
library_construction_protocol: Original and ectopic tentacles were collected 7 days after the third electroporation from mock, Zic4, Sp5, and Zic4/Sp5 RNAi animals. When possible, the proximal and distal tentacle halves were separated (original tentacles of Zic4 RNAi and Sp5 RNAi). Corresponding samples were pooled in the lysis buffer, frozen on dry ice and stored at -80 °C for RNA extraction. The RNA was extracted with the Single Cell RNA Purification Kit (Norgen), according to the manufacturer's instructions. cDNA amplification was performed using the SmartSeq2 approach as per the original protocol (31). Full length cDNA was processed for Illumina sequencing using Tagmentation with an in-house purified Tn5 transposase (32): 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).