Illumina HiSeq 2500 sequencing; RNA-seq Time Course of Developing Hydra Spheroids in Hydra Medium (HM) and isotonic medium (70mM sucrose) conditions
RNA-seq Time Course of Developing Hydra Spheroids in Hydra Medium (HM) and isotonic medium (70mM sucrose) conditions
Spheroids were collected individually in 96 well plates in 45 µl of RLT+ buffer (Qiagen) and stored at – 80 °C for a later RNA purification. Over the course of 10 h, few dozens of spheroids were prepared every hour from the 105 strain, let to close for 2 h in DM and then transferred to either HM or HM with 70 mM sucrose. 8 spheroids from each batch were then collected 3 h and 13 h after cutting the last batch, thus creating a time course spanning a window of 22 h.  RNA purification was performed using the Zymo Direct-zol MagBead reagents following a modified manufacturer protocol. Briefly, 45µl of Zymo Binding Buffer and 4µl of MagBeads were added to the samples/RLT+ buffer and incubated for 10min. Beads were washed 3 times with 100% ethanol and incubated with 12.5µl of DNAseI (Zymo) for 10min. The RNA was captured back on the beads using 100µl of MagBead PreWash buffer (10min incubation). After washing the beads 3 times with 100% ethanol, elution was performed with water (13µl) at 55C for 15min. All above incubations were performed at room temperature on a plate shaker unless specified. The resulting RNA quality was assessed using bioanalyser or tapestation RNA HS kit, and concentration determined using Qbit. 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).