Kapa library quantification kit

RNA was extracted from 1 × 10⁷ cells with the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol and the concentration was measured with a Qubit 3.0 Fluorometer. For NGS, 1 µg of RNA was treated with the Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) (Vazyme) according to the manufacturer’s protocol and the concentration was measured again. Subsequently, 10–100 ng of rRNA depleted RNA was used to prepare RNA-seq library with QIAseq Stranded RNA Library Kits (Qiagen). Size distribution of DNA fragments of final libraries were confirmed using an Agilent Fragment analyzer with the DNF 474 kit and libraries were quantified with a Qubit 3.0 Fluorometer and the KAPA library quantification kit. All the DNA libraries were pooled and sequenced on the Illumina NextSeq 500 platform.
For RT-qPCR, extracted RNA was subjected to DNase I (NEB) treatment and purified with Agencourt® RNAClean™ XP (Beckman Coulter) before first-strand synthesis. First-strand synthesis was carried out by using Superscript III Reverse Transcription System (Thermo Scientific) according to the manufacturer’s protocol. cDNA was then analyzed by qPCR on LightCycler® 480 Instrument II. Primers used in this study are listed in Supplementary Data 4.

DNA was isolated from purified virions using phenol–chloroform extraction as described previously [10 (link)]. Libraries for genome sequencing were constructed from virion DNA following the manufacturer’s protocol and reagents supplied in Illumina’s TruSeq DNA PCR-free sample preparation kit (FC-121-3001) [10 (link)]. The purified library was quantified using a KAPA library quantification kit (KK4824), and library fragment sizes were confirmed by Fragment Analyzer (Agilent, Santa Clara, CA, USA). Libraries were diluted, pooled, and sequenced using a paired-end 75-base read length according to Illumina’s standard sequencing protocol for the MiSeq. Library preparation and sequencing were conducted by the University of Missouri DNA core facility.

RNA was extracted by standard Trizol extraction techniques. Libraries were made using either NEBNext mRNA Second Strand Synthesis Module (E6111) followed by NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB-E7645) or NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760) with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NE7490) as per manufacturer’s protocols using half reactions. A total of 13 cycles of amplification was used for library enrichment; quality and size distribution of the libraries were ascertained by running on a Bioanalyzer High Sensitivity DNA Chip (Agilent (Santa Clara, USA), 5067–4626), and concentration was determined using KAPA Library Quantification Kit (KK4824). Libraries were sequenced on an Illumina HiSeq 2500 system by the Babraham Sequencing Facility. The experiment compared 3 independent replicates WT (N2) and hsf-1^neuro (AGD1289) animals.

Genomic DNA paired-end sequencing was performed for the type strain of C. jiufengensis (CBS10846), C. pseudojiufengensis (CBS10847), C. oxycetoniae (CBS10844), C. margitis (CBS14175) and C. theae (CBS12239), as described in Mixão et al.¹² (link) For C. theae a mate-pair library was also sequenced. To this end, DNA was fragmented to sizes between 1 and 20 kb using a transposase that binds biotinylated adapters at the breaking point. Strand displacement was performed to ‘repair’ the nicks left by the transposase. Fragment sizes of 3–6 kb were then selected on a 0.8% agarose gel and were then circularized. Non-circularized DNA was removed by digestion. The circular DNA was then mechanically sheared to fragments of ∼100 bp to 1 kb and the fragments containing the biotinylated ends were pulled down using magnetic streptavidin beads and submitted to a standard library preparation. A final size selection on 2% agarose gel was done and fragments of 400–700 bp were selected for the final library. Final libraries were analysed using Agilent High Sensitivity chip to estimate the quantity and check size distribution and were then quantified by qPCR using the KAPA Library Quantification Kit (ref. KK4835, Kapa Biosystems) prior to amplification with Illumina’s cBot. Libraries were sequenced 2 × 125 bp on Illumina’s HiSeq 2500.

Cell pellets were resuspended in RLB buffer and sonicated and the soluble chromatin was incubated with antibodies and Protein-G/A magnetic beads. The complexes were washed with RLB buffer, followed by ChIP buffer 1 and 2 (Active Motif), eluted with elution buffer and subjected to Proteinase-K treatment. ChIP DNA was purified using PCR DNA purification columns (Qiagen). For ChIP-Seq, MDA-MB-453 cells (5 × 10⁷ cells) were treated with vehicle or (R)-9b. Cell pellets were resuspended in RLB buffer and sonicated for 25 s. The soluble chromatin was incubated overnight at 4 °C with antibodies and protein-G/A magnetic beads [21 (link)]. Ten nanograms of immunoprecipitated DNA was used to generate sequencing libraries using the Kapa Hyper Prep Kit (Roche Sequencing Solutions Inc., Pleasanton, CA). The size and quality of the library was evaluated using the Agilent BioAnalyzer (Agilent Technologies, Inc., Santa Clara, CA), and the library was quantitated with the Kapa Library Quantification Kit. Each enriched DNA library was then sequenced on an Illumina NextSeq 500 sequencer to generate 40–50 million 75-base paired-end reads (Illumina, Inc., San Diego, CA). The raw sequence data were aligned using BowTie 2 [68 (link)], and the binding sites were identified using the MACS peak-finding software [69 (link)].