Sampling, DNA and RNA extraction and sequencing
We sampled two Balearic shearwater adults and one chick. Adults were sampled on Sa Cella colony, Mallorca (male) and on Sa Conillera, Eivissa (unsexed) in 2004, while the chick was sampled on Conills islet (Mallorca) in July 2019. From here on the animals will be referred to as male-Mll, unsexed-Ei and chick-Mll, respectively. Special permits to obtain the samples were issued by Conselleria de Medi Ambient, Agricultura i Pesca (Govern de les Illes Balears, Spain).
We extracted DNA from blood samples preserved in absolute ethanol for both adults. The DNA extraction for the male-Mll was performed with DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s instructions, and with Blood & Cell Culture DNA Mini Kit (Qiagen) for the unsexed-Ei. RNA was extracted from the chick-Mll’s blood cells preserved in RNAlater 1:5 using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocols. We performed the quality control with gel electrophoresis and NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA), and the quantification with an Invitrogen Qubit Fluorometer 2.0 (Broad Range kit).
We obtained the reference genome combining short-read and long-read sequencing libraries, and using RNA-seq data to assist with the annotation. First, an Illumina TruSeq DNA PCR Free library (insert size = 350 bp) was prepared by Macrogen (South Korea) using DNA from male-Mll, and sequenced using two HiSeq X Ten runs (2x150bp). Second, long-read libraries were prepared, from the DNA of unsexed-Ei, using the Ligation kit SQK-LSK109 1D from ONT (Oxford Nanopore Technologies) (N50 of 9431 bp) at CNAG (Centro Nacional de Análisis Genómico, Spain) and sequenced through five runs of MinION on FLO-MIN106 flow cells. Third, we prepared RNA sequencing (RNA-seq) libraries from the chick-Mll’s RNA using the TruSeq RNA Sample Prep Kit v2 with Ribo-Zero, and we sequenced the libraries on a NovaSeq 6000 (2x100bp) (Macrogen, South Korea).