The head-regeneration transcriptome of the planarian Schmidtea mediterranea
The head-regeneration transcriptome of the planarian Schmidtea mediterranea
Animals were kept at 20C in Montjuic solution (1 mM CaCl2, 0.1 mM KCl, 1.6 mM NaCl, 1.2 mM NaHCO3, 0.1 mM MgCl2, 1mM MgSO in water) and fed with veal liver once per week. For each sample, 5 planarian trunks were dissected from heads and tails. For control samples, samples were frozen immediately after dissection. The 1 and 6 hr samples were generated the following way:  At timepoint 0, the head and the tail was amputated, so there were now 3 pieces: one animal => head + trunk + tail. These 3 pieces were kept in the same culture vessel together and all allowed to regenerate. Each piece started to regenerate at each wound.  After 1 hr or 6 hrs, all three pieces (including whatever regenerated in this short period of time) were extracted together (head + trunk + tail). . Total RNA was extracted using TRIzol (Invitrogen). Strand-specific sequencing libraries were prepared from polyA RNA selected material using Applied Biosystems Whole Transcriptome Library Preparation protocol (Part number 4409491 Rev. C). In brief, 1 mg of polyA RNA was isolated using the MicroPoly(A)Purist kit (Ambion), fragmented using RNAse III (Applied Biosystems) and column purified. SOLiD sequencing adapters were ligated to the RNA RNA using components from the SOLiD Small RNA Expression Kit (Applied Biosystems). Afterwards, the RNA was reverse transcribed using ArrayScript Reverse Transcriptase and cDNA purified using the MinElute PCR purification kit (Qiagen). cDNA fragments were separated using by electropheresis on Novex 6% TBE-Urea gels, stained with SYBR Gold nucleic acid gel stain and sequences between 100-200 nt were selected. Afterwards, fragments were PCR amplified using AmpliTaq for 15 cycles to obtain templates for emulsion PCR and sequencing.
Experimental Factor: TIME
0 other
Experimental Factor: ORGANISMPART
whole_organism
Experimental Factor: BATCH
3