RNA standard methods

2.2.2.1 In vitro synthesis of capped sense mRNA

Capped sense mRNA was synthesized using the SP6 or T7 mMessage mMachine Kits (Ambion) according to manufacturer’s protocol. A reaction of 20 µl contained 1 µg of linearized plasmid and was incubated at 37°C for 2 hours.

After DNase digestion using 5 U of Turbo DNase I, the synthesized mRNA was purified using the Illustra RNAspin Mini kit (GE Healthcare) and eluted in 30 µl RNase free water at 80°C. RNA concentration was measured using the NanoDrop 2000c spectrophotometer (Thermo Scientific) and the quality was

checked on a 1% agarose gel. The RNA was aliquoted (2-4 µl) and stored at – 80°C.

2.2.2.2 In vitro synthesis of antisense RNA

For the detection of in vivo transcripts antisense RNA was used in whole mount in situ hybridizations (WMISH).

A standard reaction of 25 µl contained 1 ng linearized plasmid, 1x Transcriptionbuffer (Fermentas), 1 mM rATP, rGTP, rCTP (Boehringer), 0.64 mM rUTP (Boehringer), 0.36 mM Digoxygenin-rUTP (Boehringer), 0.03 µM DTT, 1.6 U/µl Ribolock RNase inhibitor (Fermentas), 1.2 U/µl T3, T7 or SP6 RNA-Polymerase, add RNase-free water. The reaction was incubated for three hours at 37°C, followed by template digestion with 0.2 U/µl Turbo DNaseI (Ambion) for 30 min at 37°C. The RNeasy Mini kit (Qiagen) was used to purify the synthesized antisense RNA according to manufacturer’s instructions. The RNA was eluted twice with 50 µl RNase-free water at 80°C. The quality was checked on a 1% agarose gel. The antisense RNA was stored at -20°C in 1 ml hybridization mix (see whole mount in situ hybridization).

2.2.2.3 Total RNA isolation from ectodermal explants and whole embryos

For the isolation of total RNA three embryos or 50-100 ectodermal explants were fixed in liquid nitrogen and lysated in 400 µl peqGOLD TriFast reagent (Peqlab) using a 29-gauge syringe and afterwards vortexed for 30 sec.

After addition of 80 µl chloroform (Roth) and vortexing for 30 sec, the samples were centrifuged for 10 min at 13,000 rpm and 4°C. The supernatant (200 µl) was transferred into a new eppendorf tube. 200 µl chloroform were added and the sample vortexed for 30 sec and centrifuged for 5 min at 13,000 rpm at 4°C.

The supernatant (180 µl) was transferred into a new eppendorf tube and 180 µl isopropanol were added. After vortexing, the samples were kept overnight at -20°C for precipitation. After precipitation the samples were centrifuged for 30 min at 13,000 rpm at 4°C and the pellet was washed with 400 µl 70% ethanol.

After air drying the pellet was dissolved in 12.5 µl RNase-free water and DNase digestion was carried out using DNaseI (1 U/µl) (Thermo Scientific) for 30 min at 37°C. The DNaseI was denatured by incubation at 70°C for 10 minutes and the

RNA concentration measured on the NanoDrop 2000c spectrophotometer (Thermo Scientific). The quality of the RNA was analyzed with the 2100 Bioanalyzer (Agilent). To check for genomic DNA contamination, a control PCR with H4 or ODC was performed.

2.2.2.4 Reverse transcription

For the synthesis of 10 µl cDNA, 50-75 ng RNA was used in a standard reaction mix containing 1x Go Taq flexi buffer, 5 mM MgCl2 (Fermentas), 2.5 mM random hexamer (Invitrogen), 1 mM dNTP mix (Thermo Scientific), 0.8 U/µl Ribolock RNase inhibitor (Thermo Scientific), 2 U/µl MulV reverse transcriptase (Roche). The reaction was carried out in a thermocycler. After an initial incubation for 20 min at 20°C the reaction was incubated for 1 hour at 42°C and terminated at 95°C for 5 min.

2.2.2.5 RNA-sequencing

2.2.2.5.1 Total RNA isolation from ectodermal explants

For the isolation of total RNA for RNA-sequencing 70-100 ectodermal explants were fixed in liquid nitrogen and lysates prepared in 360 µl peqGOLD TriFast reagent (Peqlab) using a 29-gauge syringe and afterwards incubated for 10 min at RT. After addition of 72 µl chloroform the solution was incubated at RT for 5 min and centrifuged for 20 min with 13,000 rpm at 4°C. The upper phase was transferred into a new Eppendorf tube to which 200 µl chloroform was added. The solution was centrifuged for 10 min at 4°C and the upper phase transferred into a new Eppendorf tube. After adding 180 µl isopropanol the RNA was precipitated overnight at -20°C. The next day, the solution was centrifuged for 30 min at 4°C. The pellet was washed with 70% ethanol and centrifuged for 5 min at 4°C. The pellet was air dried, and resuspended in 43.5 µl RNase-free water. For the digestion of genomic DNA, the RNA was incubated for 1 h at 37°C in a 50 μl reaction containing 1x DNaseI reaction buffer (Thermo Scientific), 1 μl DNaseI (Thermo Scientific) and 0.5 μl RNase inhibitor (Thermo Scientific). To stop the DNase treatment, 150 µl RNase free water and 200 µl phenol/chloroform/isoamylalcohol (25/24/1, Roth) was added to the RNA. After centrifugation for 10 min at 4°C the upper phase was transferred to a new

Eppendorf tube, 1 vol. of chloroform/isoamylalcohol (24/1) was added.

Afterwards the RNA was centrifuged for 10 min at 4°C. The supernatant was transferred to a new Eppendorf tube to which 1/10 vol. of 5 M ammonium acetate and 1 vol. of isopropanol was added. The solution was incubated overnight at -20°C. The following day the RNA was centrifuged for 30 min at 4°C and the pellet washed twice with 70% ethanol and centrifuged for 5 min at 4°C. The pellet was air dried and resuspended in 20 µl RNase free water. The quality of the RNA was analyzed with the 2100 Bioanalyzer (Agilent). To check for genomic DNA contamination, a control PCR with H4 or ODC was performed.

2.2.2.5.2 Sample preparation and sequencing

LIbrary preparation and sequencing was performed by the DNA Microarray and DeepSequencing Facility Göttingen (UMG) -Transkriptomanalyselabor (TAL). Before sequencing, the RNA-libraries were prepared with the "TruSeq RNA Sample Prep Kit v2" (Illumina) following the manufacturer’s protocol. The single read (50 bp) sequencing was performed using the HiSeq 2000 (Illumina). Three independent biological replicates were used for each sequencing reaction. The quality of the sequencing was

controlled using the FastQ software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).

2.2.2.5.3 Sequencing alignment (performed by TAL)

The sequence reads were aligned to the genome reference sequence of X. tropicalis (Joint Genome Institute assembly v4.2) using the STAR alignment software version 2.3.0e (Dobin et al., 2012). For the alignment, 5 mismatches were allowed within 50 bases.

2.2.2.5.4 Statistical analysis (performed by TAL)

For normalization and identification of differentially expressed genes the R/Bioconductor environment (www.bioconductor.org) with the DESeq2 package version 1.2.10 (Anders and Huber, 2010) was used. Genes showing at least a 2-fold change and a FDR-corrected p-value < 0.05 were considered as candidate genes.

2.2.3 X. laevis embryo culture and micromanipulations