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. Asexual planarians of the species Schmidtea mediterranea were starved for one week prior to decapitation with a razor blade. Regeneration was allowed to occur for 16 different periods of time (30 min, 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 18, 24, 36, 48, 72h). A second cut anterior to the pharynx (pre-pharyngeal cut) was performed to separate the anterior regenerating part from the rest of the body. 8 planarian pre-pharyngeal pieces from similar time points were pooled (Library 1: 30 min and 1h, four animals each; Library 2: 2h and 3h, four animals each; Library 3: 4, 5, 6, and 8h, two animals each; Library 4: 10, 12, 16, and 18h, 2 animals each; Library 5: 24, 36, 48, and 72h, 2 animals each), and immediately deep-frozen. 16 non-regenerating pieces served as two independent biological replicates (8 animals each; Library 6 and 7). Samples were processed in two batches: material from control Library 6 was processed together with Libraries 1-3, while contrl Library 7 was processed together with Libraries 4 and 5. Total RNA was extracted from planarian fragments using Trizol. 0.5 microgram of polyA RNA was isolated from total RNA using the MicroPoly(A)Purist kit (Ambion), fragmented and precipitated. First strand cDNA was synthesized using SuperScript II (Invitrogen) and the second strand was added using RNAseH and DNA Polymerase I. cDNA was purified with the QIAquick PCR purification kit (QIAGEN) and ends were repaired with Klenow DNA polymerase. The 3 prime ends were adynlated with Klenow exo (3 prime to 5 prime exo minus) enzyme and ligated to the sequencing adapters. cDNA templates were separated by Agarose gel electrophoresis and ca. 200 bp sequences were selected. Templates for sequencing were PCR amplified using Phusion DNA polymerase (Finnzymes Oy) and purified with the QIAquick PCR purification kit (QIAGEN).
Experimental Factor: TIME
10 to 18 other
Experimental Factor: ORGANISMPART
pre-pharyngeal fragment, regenerating the head
Experimental Factor: BATCH
2