Agilent ngs workstation

Total RNA was extracted using the RNeasy Fibrous Tissue Mini Kit (#74704, Qiagen). RNA libraries for sequencing were prepared using poly-A selection and the Illumina RNA strand-specific TruSeq Stranded mRNA Sample prep kit with 96 dual indexes (Illumina, CA, USA) according to the manufacturer’s instructions with the following changes. The protocols were automated using an Agilent NGS workstation (Agilent, CA, USA) using purification steps as described^{80 (link),81 (link)}.
Clustering was done by ‘cBot’ and samples were sequenced on HiSeq2500 (HiSeq Control Software 2.2.58/RTA 1.18.64) with a 2 × 125/2 × 150 setup using ‘HiSeq SBS Kit v4’ chemistry. The Bcl to FastQ conversion was performed using bcl2fastq-v2.17.1.14 from the CASAVA software suite. The quality scale used was Sanger/phred33/Illumina 1.8+.

RNA libraries were prepared using the TruSeq Stranded mRNA sample prep kit with 96 dual indexes (Illumina, CA, USA) in accordance with the manufacturer’s instructions except for the following changes: The protocols were carried out automatically on an Agilent NGS workstation (Agilent, CA, USA) using the purification steps described by Lundin et al.54 (link) and Borgström et al.55 (link). After clustering on cBot, the samples were sequenced on a HiSeq 2500 as recommended by the manufacturer. Demultiplexing and conversion were performed with CASAVA v1.8.2. software (Illumina, CA, USA) employing the Sanger/phred33/Illumina 1.8+ quality scale. On average, each run generated 44.3 million paired-end reads per sample, of which 81% mapped to the human genome. For 5 samples, the sequencing quality was low and these were excluded from further analysis, leaving 62 samples from patients and 62 from healthy controls.

Total RNA was extracted using the RNeasy Fibrous Tissue Mini Kit (#74704, Qiagen). RNA libraries for sequencing were prepared using poly-A selection and the Illumina RNA strand-specific TruSeq Stranded mRNA Sample prep kit with 96 dual indexes (Illumina, CA, USA) according to the manufacturer’s instructions with the following changes. The protocols were automated using an Agilent NGS workstation (Agilent, CA, USA) using purification steps as described^{56 (link),57 (link)}.
Clustering was done by ‘cBot’ and samples were sequenced on HiSeq. 2500 (HiSeq Control Software 2.2.58/RTA 1.18.64) with a 2 × 125/2 × 150 setup using ‘HiSeq SBS Kit v4’ chemistry. The Bcl to FastQ conversion was performed using bcl2fastq-v2.17.1.14 from the CASAVA software suite. The quality scale used was Sanger/phred33/Illumina 1.8 + .

Total RNA was extracted from the EMs and suspensors using RNAqueous Micro kit (Ambion). RNA quality was assessed in terms of RNA integrity number (RIN) by Bioanalyzer (Agilent 2100 expert). To obtain sufficient amount of RNA required for RNA-seq, the RNA extracted from the embryos of cell line 11:18 was subsequently amplified using MessageAmpIIaRNA Kit (Ambion).
cDNA library preparation and subsequent sequencing were performed at the SciLifeLab (Stockholm, Sweden). Strand-specific cDNA libraries were prepared with TruSeq Stranded mRNA Sample prep kit of 96 dual indexes (Illumina, CA, USA) according to the manufacturer’s instructions except for the following changes. The protocols were automated in Agilent NGS workstation (Agilent, CA, USA) using purification steps as previously described [15 (link), 16 (link)]. Clonal clusters were generated using cBot (Illumina) and sequenced on HiSeq2500 (Illumina) according to manufacturer's instructions. Bcl to Fastq conversion was performed with bcl2Fastq v1.8.3 from the CASAVA software suite. The quality scale was Sanger / phred33 / Illumina 1.9. The obtained data were deposited to the European Nucleotide Archive (ENA) and is accessible under the accession number PRJEB22154.