Study participants
Patients with mild-to-moderate COVID-19 admitted to our COVID-19 subunit at Nishi-Kobe Medical Center within six days after the disease onset were eligible for inclusion between August 2020 and February 2021. SARS-CoV-2 infection was confirmed by RT-qPCR analysis. Also, blood for RNA-seq analysis was collected at admission. Disease severity was evaluated using the WHO definition (meet any of the followings: oxygen saturation < 90%, respiratory rate > 30 breaths/min, and signs of severe respiratory distress or not). Based on the subsequent clinical courses, patients were grouped into two groups, deteriorated (developed severe diseases) and nondeteriorated (had only mild-to-moderate diseases). The baseline characteristics of patients with COVID-19, which include age, sex, the day of disease onset and sample collection, body temperature, pulse rate, SpO2, white blood cells, neutrophils, lymphocytes, monocytes, CRP, LDH, and D-dimer were extracted from medical records. This study was conducted following the World Medical Association’s Declaration of Helsinki and approved by the ethical review board of Nishi-Kobe Medical Center (#2021–22). Informed consent was obtained from all study participants.
PBMCs isolation and total RNA extraction
Density gradient centrifugation was used to prepare PBMCs. In detail, Lymphoprep (STEMCELL TECHNOLOGIES, Canada) was added to blood samples and poured into SepMate(STEMCELL TECHNOLOGIES), followed by dilution using an equal volume of saline. After centrifugation at 1,200 g for 10 min, the layer containing PBMCs was transferred into a new tube and washed with saline. Then, the pellet was collected by centrifugation at 300 g for 8 min. Total RNA was extracted using Maxwell RSC instrument and Maxwell RSC simplyRNA Blood Kit (Promega, WI, USA) according to the manufacturer’s instructions and stored at −80 °C for future analysis.
RNA-seq analysis
Total RNA from six deteriorated and six nondeteriorated patients with COVID-19 were analyzed. Libraries were prepared with the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) and subjected to sequencing with an Illumina Novaseq 6000 instrument (Illumina). Reads were trimmed using FASTQ groomer30 and aligned to the GENCODE human reference genome GRCh37 (hg19, GENCODE website) using the HISAT2 function31 on the Galaxy platform32. Gene transcripts were quantified using DESeq233, and the volcano plot was generated by ggplot2 on Galaxy. Heatmap was generated by ClustVis34. The enrichment of DEGs compared with the deteriorated and nondeteriorated group was analyzed using ShynyGO35 and GSEA36. In silico protein–protein interaction analysis was conducted using STRING37. RNA-seq data have been deposited on GEO and can be accessed at the following link.
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE207625
RT-qPCR analysis
RT-qPCR was performed using iTaq Universal SYBR Green One-Step Kit (BIO-RAD Laboratories, CA, USA) according to the manufacturer’s instructions. Each measurement was duplicated using a CFX96 Real-Time System (BIO-RAD). The assay conditions were: 50°C for 10 min for reverse transcription, and 95°C for 1 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Primer pairs were designed using PrimerBank (http://pga.mgh.harvard.edu). b-actin (forward, 5′-CATGCCATCCTGCGTCTGGA-3′, reverse, 5′-CCGTGGCCATCTCTTGCTCG-3′), HIST1H2BO (forward, 5′-GACCCGGCTAAATCTGCTCC-3′, reverse, 5′-GGCCTTGGTTACGGCTTTC-3′, reverse, 5′-GGCCTTGGTTACGGCTTTC-3’), HIST1H2AE (forward, 5′-CTACTCCGAACGAGTCGGG-3′, reverse, 5′-GATGGTCACGCGACCTAGAAG-3′) were used.
Statistical analysis
All data represent mean ± standard deviation (SD). The normality of the data was evaluated by Kolmogorov–Smirnov normality test. Statistical significance was determined by a two-sided unpaired t-test or Mann–Whitney test. A p value of < 0.05 was considered significant.