4150 tapestation system

Three biological replicates were collected for each group (LPS + I1 and LPS) for RNA isolation. Cells were lysed, and RNA was isolated using an RNeasy Plus mini kit (QIAGEN) according to the manufacturer’s instructions for cell culture. The number of cells used per sample ranged from 1 × 10⁶ to 5·× 10⁶. RNA was quantitated using a Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and a Qubit RNA HS assay kit, following the manufacturer’s instructions. The RNA integrity number (RIN) for each sample was measured with a TapeStation 4150 system (Agilent, Santa Clara, CA, USA) using the high-sensitivity RNA ScreenTape. The RIN values for all samples were between 7 and 9.5. From this total RNA, RNA sequencing (RNA-Seq) was performed for the transcriptomics assay, followed by quantification of the expression of different genes using qPCR.

A total of 111 representatives of C. jejuni isolates from chicken meats at regions B, C and E were arbitrarily selected to include highly contaminated sample origin (>3.0 logCFU/g). The bacterial isolates were grown on Mueller–Hinton agar (Becton Dickinson) at 37°C for 20 h under microaerophilic conditions using the AnaeroPack-MicroAero system, followed by centrifugation at 4,000× g for 5 min. Accordingly, we extracted DNA from the bacterial pellets using the Maxwell Blood DNA Kit (Promega, Madison, WI, USA). We measured the concentration and quality of the extracted DNA on a TapeStation 4,150 system (Agilent Technologies, Santa Clara, CA, USA). The samples were stored at −80°C until further use.

TCR repertoires of FACS-sorted Tax_301-309-CTLs (approximately 0.5-8.5 x10⁴ cells) and CD8⁺ T-cells (approximately 1.5-6.3 x10⁵ cells) in PBMCs from eleven HAM patients (HAM-1, -4, -5, -7, -8, -9, -11, -12, -13, -14, and -15) and CSF whole cells (approximately 0.8-2.7 x 10⁴ cells) of four HAM patients (HAM-8, -9, -11, and -12) were analyzed. The total RNA of each sample was independently extracted using the RNeasy Micro kit (Qiagen, Valencia, CA). Then, cDNA was amplified using iRepertoire human TCRβ kits (iRepertoire, Huntsville, AL, USA) according to the manufacturer’s protocol. The quality (size and integrity) and quantity (concentration) of the final library for sequencing were checked by the TapeStation4150 system (Agilent Technologies, Santa Clara, USA) and Qubit 4.0 fluorometer (Thermo Fisher Scientific), respectively. Sequencing was performed using MiSeq platform (Illumina, San Diego, CA, USA) with 250 bp paired-end reads. The data were analyzed in a provided pipeline by iRepertoire (http://www.irepertoire.com). The illustrative tree map was used to represent each unique T-cell clone. The sequence run data including reads, total CDR3, and distinct CDR3 have been summarized in Supplementary Table 1.

Illumina sequencing libraries were prepared from unselected genomic DNA (WGS) and from the same fractions F2, F3 and F4 that were analyzed by Southern blot. After shearing to an average fragment size of 250 bp with a Covaris ME220 Sonicator, libraries were prepared with Kapa Hyper Prep kit (Roche) according to the manufacturer’s instructions with 12 amplification cycles then they were sequenced on an Illumina NovaSeq 6000 using paired-end 100x100 as sequencing mode.
Nanopore sequencing was performed from fractions derived from an independent digestion and sucrose gradient experiment. Before preparation of libraries for Nanopore sequencing, fractions F4 to F6 were pooled and 9 μg of this DNA was treated with Short Read Eliminator kit (cutoff <25 kb, Circulomics cat# SKUSS-100-101-01) to further remove contamination from shorter DNA fragments. Libraries were prepared from this sample, from fraction F3 and from total genomic DNA (WGS) using the Library Preparation by Sequencing kit (Oxford Nanopore Technology). For all samples, sequencing was performed on a Spot-ON Flow Cell (R9.4.1) on a MinION Mk1B device.
Libraries were quantified with Qubit dsDNA HS Assay Kit (Thermo Fisher) and checked by capillary electrophoresis with a TapeStation 4150 system (Agilent).

All samples were prepared in biological triplicates for V-K562, B-K562 and G-K562 cells harvested at 0.7–0.8 × 10⁶ cells/mL (Loats et al., 2021 (link); Toska et al., 2012 (link)). Cells taken for ATAC-seq were treated with DNase and incubated for 30 mins buffered with 25mM MgCl2 and 5mM CaCl2 prior to harvesting. Samples of 2 × 10⁶ for RNA-seq and of 1 × 10⁵ cells for ATAC-seq were then pelleted and snap-frozen in liquid nitrogen until library preparation. Total RNA was isolated from the cells using the RNeasy Mini Kit (Qiagen, cat 74104), RNA Integrity Number (RIN) was determined for each sample using Tape Station 4150 System (Agilent). Libraries were prepared according to manufacturer’s instructions for TruSeq Stranded mRNA Library kit (Illumina, cat #20020594) and ATAC-Seq Kit (Active Motif, cat #53150). Libraries were sequenced on Illumina NextSeq 500 in 75-nt (RNA-seq) and 42-nt (ATAC-seq) experiments in paired-end mode.

RNA from the viral infected and uninfected cells (six time points, three biological replicates), as well as from the CHX-treated samples, were purified using the spin column-based NucleoSpin RNA kit (Machery-Nagel, Bethlehem, PA, USA), as described in the manual. The following modifications were carried out: (1) proteinase K (0.37 mg/mL final concentration) was added to the samples at the lysis step; (2) TURBO DNA-free™ Kit (Invitrogen) was used to eliminate the potential residual genomic DNA from the isolated RNA samples. The RNA concentration was determined by using Qubit 4.0 Fluorometer and Qubit RNA BR (Broad-Range) Assay Kit (Invitrogen). The quality of RNA was assessed based on RIN values using TapeStation 4150 system (Agilent). RIN scores ≥ 9.3 were used for sequencing.