Truseq stranded rna protocol

Total RNA was extracted from zebrafish embryos at 80% epiboly by Trizol extraction and gDNA was genotyped for tcf7l1a to identify wildtype, heterozygous and homozygous embryos. RNA from six wild-type and six tcf7l1a^-/-mutant embryos was DNase treated for 20 min at 37°C followed by addition of 1 ml 0.5M EDTA and inactivation at 75°C for 10 min to remove residual DNA. RNA was then cleaned using 2 volumes of Agencourt RNAClean XP (Beckman Coulter) beads under the standard protocol. Stranded RNA-seq libraries were constructed using the Illumina TruSeq Stranded RNA protocol with oligo dT pulldown. Libraries were pooled and sequenced on two lanes of Illumina HiSeq 2000 in 75 bp paired-end mode. Sequence data were deposited in ENA under accession PRJEB9957. FASTQ files were aligned to the GRCz11 reference genome using TopHat (v2.0.13, options: --library-type fr-firststrand, Kim et al., 2013 (link)). The data were assessed for technical quality (GC-content, insert size, proper pairs etc.) using QoRTs (Hartley and Mullikin, 2015 (link)). Counts for genes were produced using htseq-count (v0.6.0 options: --stranded=reverse, Anders et al., 2015 (link)) with the Ensembl v93 annotation as a reference. Sequence data were deposited in ENA under accession PRJEB9957. Differential gene expression was analysed using DESeq2 (Love et al., 2014 (link)).

RNA sequencing was performed on three biological replicates for the control and four biological replicates for Lmx1a/b cKO mice. For each sample, two technical replicates were performed. For each E15.5 embryo used, VTA and SNpc were dissected from eight antero–posterior levels across the entire DA domain as revealed by TH immnostaining. Following RNA extraction, RNA-seq libraries were constructed using the Illumina TruSeq Stranded RNA protocol with oligo dT pulldown and sequenced on Illumina HiSeq2500 by 150-bp paired-end sequencing. One sample was excluded before the sequencing because the amount of RNA was too low to measure the RNA quality. Reads were aligned using STAR aligner (version 2.4.2a) to the mouse genome assembly GRCm38. Raw read counts per gene were obtained using featureCount in the R package Rsubread with the GENCODE annotation vM8. The gene differential expression analysis was done using DeSeq2^{50 (link)}. We only considered genes with >10 reads across all of the samples. A total of 225 genes with adjusted p value <0.2 were reported as being significantly differentially expressed. Data are available at Array Express under accession number E-MTAB-5986.

Prior to RNA isolation, the anode slices and liquid cultures were stored at −20°C in LifeGuard™ Soil Preservation Solution according to the manufacturer‘s instructions (MoBio; USA). After thawing, the cells were loosened gently from the anode surface using a cell mill (MM400, Retsch; Germany) for 30 min at 8 Hz. RNA was isolated with the RNA Power®Total RNA Isolation Kit (Qiagen; Germany) according to the manufacturer's instructions. DNA was removed with the DNA-free™ Kit (Thermo Fischer; USA) at 37°C. Sequencing libraries were prepared from 400 ng of total RNA samples following the TruSeq stranded RNA protocol (Illumina; USA, without purification). Sequencing was performed on a HiSeq1500 using SBS v3 kits (Illumina) generating paired-end reads of 2 × 50 nucleotides. Cluster detection and base calling were performed using RTA v1.13 (Illumina). The read quality was evaluated with CASAVA v1.8.1 (Illumina). 255 million reads were generated via sequencing with 93% of them having a quality Phred score of Q30 or more.
For the isolation of 16S rRNA, 1 ml of a liquid culture was harvested and the pellet was resuspended in 0.9% NaCl and heated for 5 min at 90°C. The 16S rRNA genes were amplified using this suspension as template and the primers Bak27F and BakUniversal1492R (Supplementary Table 1) Sequencing was carried out by GATC (GATC Biotech AG, Konstanz).