Transcriptomic analysis of PBMC in long covid
Following isolation, RNA purity and integrity were respectively assessed using a NanoDropTM spectrophotometer (Thermo Scientific, Nanodrop 2000c) and RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Samples with RNA integrity number values >7.5 were retained for further processing. Before library preparation, mRNA and non-coding RNAs (lncRNAs) were enriched by MGIEasy rRNA Depletion Kit (MGI Tech, China) and DNase I treatment (NEB) was carried out for complete removal of the DNA. RNA libraries was constructed from 500 ng RNA using the MGIEasy RNA Library Prep Kit V3.0 protocol (MGI, Shenzen, China) according to the manufacturers instruction. Firstly, enriched RNA samples were fragmented in fragmentation buffer, then short fragments were subjected to reverse transcription and second-strand synthesis. The cDNA fragments were treated with standard library generation steps; end-repair, A-tailing and adapter ligation. After purification with DNA clean beads, we enriched adapter-ligated fragments using 14 PCR cycles and subjected to the following denaturation and single-strand circularization process to generate a single-stranded circular DNA library. This libraries were then used to generate DNA nanoballs (DNBs) by rolling circle replication (RCR). The resulting DNBs were then loaded into the patterned nanoarrays and sequencing reaction was performed in a DNBSEQ-G400 sequencer with a pair-end read length of 100 bp.