GSM4173975: Control larvae 1; Danio rerio; RNA-Seq
Samples were snap frozen at -80°C, then 100 μl of RNA protect bacteria reagent (Qiagen) was added, followed by mechanical trituration with a pestle blender. Samples were supplemented with 100 μl of 30 mM Tris-HCl/1 mM EDTA solution at pH = 8, 33 μl of 50 mg/ml lysozyme (Thermo Scientific), 33 μl of proteinase K >600 U/ml (Thermo Scientific) and shaken for 20 min RT. Lysis was completed by adding 700 μl of RTL buffer (Qiagen), 3 μl of 1 M dithiothreitol (Sigma-Aldrich) and mechanical disruption. Undigested debris were spun down 3 min 10000 rpm and the supernatant was supplemented with 500 μl of 100% ethanol prior loading onto RNeasy mini columns (Qiagen). From this step onwards, the manufacturer's guidelines were followed for RNA purification. RNA quality and integrity were assessed by using NanoDrop and Non-denaturing agarose gel electrophoresis. For further quality check, RNA sequencing, library construction and reads count, samples were outsourced to Vertis Biotechnologie AG (Freising, Germany). Bacterial and host mRNA were enriched prior library preparation by using Ribo-Zero Gold rRNA Removal Kit (Epidemiology, Illumina, San Diego, California). The rRNA depleted RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase . The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). For Illumina NextSeq sequencing, the samples were pooled in approximately equimolar amounts. The cDNA pool was size fractionated in the size range of 200 – 550 bp using a preparative agarose gel. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The cDNA pools were sequenced on an Illumina NextSeq 500 system using 75 bp read length.
GEO
GSM4173975
GEO Accession
GSM4173975