Nebnext multiplex oligos

In situ Hi-C was performed as previously described^{9 (link)} with slight modifications. Cells at 80% confluence in 10 cm dish were fixed in 1% pFA at room temperature for 10 min and subjected to the in situ Hi-C procedure. After proximal ligation, ligation products were enriched by centrifugation at 10,000 rpm at 4 °C for 10 min. The pellet was suspended with proteinase K solution consisting of 10 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 2 mg/ml proteinase K (Thermo Fisher Scientific) and incubated at 37 °C for 1 h, followed by overnight incubation at 68 °C with 0.5 M NaCl. DNA was purified by phenol/chloroform extraction and sonicated at 4 °C using Bioruptor (Diagenode) for 15 min by repeating the cycle of 30-sec ON and 1-min OFF. Biotin-labeled DNA was purified using Dynabeads (MyOne Streptavidin T1, Thermo Fisher Scientific). Sequencing adapters were ligated using NEBNext Ultra Ligation module (New England Biolabs, Inc) on Dynabeads, and DNA was PCR-amplified for 8–14 cycles using NEBNext Q5 Hot Start HiFi PCR master Mix and NEBNext multiplex oligos (New England Biolabs, Inc). PCR products were sequenced on Illumina NextSeq 500 platform to obtain 76-bp paired-end reads.

The genomic DNAs of A. viridinutans and A. pseudoviridinutans were extracted from a 2-d-old culture using phenol-chloroform and NucleoBond buffer set III (TaKaRa, Shiga, Japan). The DNA was fragmented in an S2 sonicator (Covaris, MA, USA), and then purified using a QIAquick gel extraction kit (Qiagen, CA, USA). A paired-end library was constructed using an NEBNext Ultra DNA Library Prep Kit (New England BioLabs, MA, USA) and NEBNext Multiplex Oligos (New England BioLabs) in accordance with the manufacturer's instructions. Mate-paired libraries with insert sizes of 3.5–4.5, 5–7, and 8–11 kb were generated using the gel selection-based protocol of the Nextera Mate Pair Kit (Illumina, San Diego, CA, USA) and a 0.6% agarose gel, in accordance with the manufacturer's instructions. The quality of the libraries was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Paired-end sequencing (100 bp) was performed by HiSeq 1500 (Illumina) using HiSeq reagent kit v1, in accordance with the manufacturer's instructions; 150-bp paired-end sequencing on a HiSeq X system (Illumina) was carried out by GENEWIZ (South Plainfield, NJ, USA).

Total RNA was extracted and purified from E14.5 fetal liver using the Qiagen RNeasy Mini Kit (Qiagen, 74104) according to manufacturer’s instructions. Polyadenylated messenger RNA (mRNA) was selectively enriched using Invitrogen Dynabead mRNA Purification Kit (Invitrogen, 61006). A sequencing library was constructed with NEBNext Ultr II RNA Library Prep Kit (NEB, E7770S) and amplified with NEBNext Multiplex Oligos (NEB, E7335S). Paired-end reads were obtained with Illumina HiSeq2500. Paired-end reads were aligned to the mouse genome (Gencode vM14) with Tophat and transcripts were assembled with Cufflinks^{64 (link)}.

Amplicon libraries were prepared as previously described [45 (link)]. Briefly, CHIKV RNA isolated from bodies and saliva was amplified by nine separate RT-PCR reactions using a high-fidelity RT-PCR kit and the primers described in Riemersma et al., 2019 [45 (link)]. The nine overlapping cDNA amplicons spanning from the 5’ to 3’ untranslated regions (UTR), excluding the first 14 nucleotides of the 5’ UTR and the last 55 nucleotides of the 3’ UTR, were pooled at equimolar ratios and enzymatically fragmented to approximately 150 bp. Illumina sequencing libraries were prepared with the fragmented cDNA using a NEBNext Ultra II DNA library prep kit and NEBNext Multiplex Oligos (New England Biolabs). Libraries of P4 infectious plasmid DNA with (pY_PCR) and without (pY) RT-PCR amplification, and P4 in vitro transcribed RNA were used as sequencing controls. An unrelated library (WNV cDNA) was also included as a control for index hopping with Illumina HiSeq 4000 sequencing [56 ]. All libraries were sequenced in parallel with paired-end 150 reads on a single flow cell lane of an Illumina HiSeq 4000 instrument at the UC Davis DNA Technologies Core. Raw fastq files are available from NCBI Sequence Read Archive under BioProject entry PRJNA541092.

All sequencing experiments were carried out in at least biological triplicate. Sequencing samples were indexed using NEBNext Multiplex Oligos (NEB). The average size and concentration of all sequencing libraries was analysed using a Bioanalyser High Sensitivity DNA Kit (Agilent) followed by qPCR using SensiMix SYBR Green mastermix (Bioline) and KAPA Illumina DNA standards (Roche). Libraries were sequenced using a NextSeq500 (Illumina). ChIP and TT-seq libraries were sequenced with 40 bp paired-end reads and RNA-sequencing libraries with 80 bp paired-end reads.