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SUPPLEMENTARY MATERIALS and METHODS PBMC Transcriptomics BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut SUPPLEMENTARY MATERIALS AND METHODS PBMC transcriptomics identifies immune-metabolism disorder during the development of HBV-ACLF Contents l Supplementary methods l Supplementary Figure 1 l Supplementary Figure 2 l Supplementary Figure 3 l Supplementary Figure 4 l Supplementary Figure 5 l Supplementary Table 1 l Supplementary Table 2 l Supplementary Table 3 l Supplementary Table 4 l Supplementary Tables 5-14 l Supplementary Table 15 l Supplementary Table 16 l Supplementary Table 17 Li J, et al. Gut 2021;0:1–13. doi: 10.1136/gutjnl-2020-323395 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut SUPPLEMENTARY METHODS Test for HBV DNA The levels of HBV DNA were detected using real-time PCR with a COBAS® AmpliPrep/COBAS® TaqMan 48 System (Roche, Basel, Switzerland) and HBV Test v2.0. Criteria for diagnosing cirrhosis Pathology The gold standard for the diagnosis of cirrhosis is a liver biopsy obtained through a percutaneous or transjugular approach.1 Ultrasonography was performed 2-4 hours before biopsy. Liver biopsy specimens were obtained by experienced physicians. Percutaneous transthoracic puncture of the liver was performed according to the standard criteria. After biopsy, patients were monitored in the hospital with periodic analyses of haematocrit and other vital signs for 24 hours. Cirrhosis was diagnosed according to the globally agreed upon criteria.2 Cirrhosis is defined based on its pathological features under a microscope: (a) the presence of parenchymal nodules, (b) differences in liver cell size and appearance, (c) fragmentation of the biopsy specimen, (d) fibrous septa, and (d) an altered architecture and vascular relationships. Depending on the size of the nodules, three macroscopic types have been defined: micronodular, macronodular, and mixed cirrhosis. Endoscopy Gastroscopy (endoscopic examination of the oesophagus, stomach, and duodenum) was performed to exclude the possibility of oesophageal and gastric varices. Radiology Ultrasound is routinely used to evaluate cirrhosis.3 The liver size was calculated from the longitudinal diameter of the right and left lobes and considered normal in the range of 9-14 cm and 7-10 cm, respectively.4 Liver morphology was based on the caudate/right lobe ratio, as the former tends to be enlarged and the latter to shrink with the progression of cirrhosis. This ratio was considered normal at a value less than 0.6.5 Liver boundaries were examined on the inferior surface of the left and right lobes, particularly in relationship with the gallbladder Li J, et al. Gut 2021;0:1–13. doi: 10.1136/gutjnl-2020-323395 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut and right kidney, and were distinguished as normal, rounded and nodular.6 Liver echogenicity was classified as normal, with increased reflectivity, and coarse and irregular in relation to the echogenicity and the distribution of parenchymal echoes.7 Portal vein diameter was measured as the largest anteroposterior diameter at the crossing point with the hepatic artery during suspended respiration and considered normal at a value less than 12 mm.8 The mean flow velocity of the portal vein was calculated using the pulsed Doppler technique9 after positioning the sample volume at the cross point of the vein with the hepatic artery and considered normal at values greater than 15 cm/s. Spleen size was assessed by the longitudinal cross-sectional area, as this parameter has been reported to correlate with the actual spleen volume10 and is considered normal at values up to 45 cm2. A small and nodular liver may be detected in patients with advanced cirrhosis, along with increased echogenicity and irregularly appearing areas. Other findings suggestive of cirrhosis in imaging are an enlarged caudate lobe, widening of the liver fissures and enlargement of the spleen. Other radiological tests include elastography techniques,11 abdominal CT and liver/bile duct MRI (MRCP). Sample preparation for mRNA sequencing For mRNA-seq, PBMCs were collected from patients and volunteers using Ficoll-PaqueTM PLUS medium (GE Healthcare, Uppsala, Sweden), and total RNA was extracted using TRIzol reagent (Ambion, Carlsbad, CA). Sequencing libraries were prepared according to the manufacturer’s instructions (TruSeq® RNA LT Sample Prep Kit v2, Illumina, San Diego, CA), including purifying and fragmenting mRNA, synthesizing first-strand cDNAs, synthesizing second-strand cDNAs, performing end repair by adenylating the 3' ends, ligating adapters, and enriching DNA fragments. The pooled library consisted of sequences with lengths of approximately 250 nucleotides. The library was sequenced using a HiSeq 2500 sequencing Li J, et al. Gut 2021;0:1–13. doi: 10.1136/gutjnl-2020-323395 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut system (Illumina). qRT-PCR qRT-PCR validation with samples from patients and experimental rats was performed to confirm the results of the transcriptomic analysis. qRT-PCR was performed using a two-step protocol with specific primers, TB Green DYE II (Takara, Beijing, China), and an ABI 7500HT instrument (Thermo Fisher, Waltham, MA) according to the manufacturer’s instructions. The amount of cDNA was optimized to ensure that the amplification of both control genes and cDNAs of interest occurred in the exponential phase. Transcripts were quantified using the comparative Ct method and normalized to the levels of the endogenous control (glyceraldehyde phosphate dehydrogenase, GAPDH). IHC The expression of potential proteins encoded by the target genes were validated using IHC in liver tissues derived from patients with HBV-ACLF and patients with LC (n=5/group) according to a previously described protocol.12 Liver specimens were obtained from patients with HBV-ACLF who received a liver transplant, while liver specimens were obtained from patients with LC during biopsies. As a control, normal liver tissues were obtained from therapeutic partial hepatectomies that were performed in patients with trauma-induced liver rupture and who exhibited no signs of hepatic dysfunction, hepatitis virus (including A-E) infection or hepatic cancer. Because the isolation of liver tissue samples from patients with stable CHB and ACHD was difficult, IHC was not performed on samples from patients with CHB and ACHD. Experimental animals Li J, et al. Gut 2021;0:1–13. doi: 10.1136/gutjnl-2020-323395 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut SPF-grade male Sprague-Dawley rats (80 g body weight) were housed under controlled environmental conditions (22±1°C, 55±5% relative humidity, 12 hour light–dark cycle) with free access to food and water ad libitum. Animal care and all experimental procedures were approved by the Animal Care Ethics Committee of the First Affiliated Hospital, Zhejiang University, and conducted in accordance with the National Institute of Health (NIH) guidelines. Establishment of the ACLF model Rats were randomly allocated into two groups (control group: n=10; ACLF group: n=30). In the ACLF group, rats were intraperitoneally administered porcine serum (Gibco, Grand Island, NY, USA) at 2 mL/kg13 twice a week for 12 consecutive weeks to generate liver cirrhosis. Afterwards, rats with liver cirrhosis were intraperitoneally injected with D-galactosamine (D- Gal) and lipopolysaccharide (LPS) in combination (800 mg/kg D-Gal and 100 µg/kg LPS) to induce acute liver failure based on chronic liver cirrhosis14, 15. Meanwhile, rats in the control group were only administered normal saline. Animals were sacrificed at 0 and 48 hours, and blood and tissue samples were collected. Evaluation of biochemical function Biochemical tests of liver function were evaluated by analysing 6 markers in rats in the control, LC and ACLF groups using an automatic clinical chemistry analyser (AU5800, Beckman, Jersey City, NJ, USA). Haematoxylin-eosin (H&E) and Masson’s trichrome (M&T) staining Liver tissues from the rat model of different stages of ACLF were collected for H&E and M&T staining to observe the pathological features and progression of liver cirrhosis. Each liver tissue section was heat-fixed at 60°C for 1 hour and then stained with H&E, as previously described.16 Li J, et al. Gut 2021;0:1–13. doi: 10.1136/gutjnl-2020-323395 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut M&T staining was performed according to recommended protocols (Maixin Biotech, Fuzhou, China and Solarbio LIFE SCIENCE, Beijing, China), as previously described.17 Identification of DEGs Adaptors and low-quality reads were removed using Trimmomatic v0.36 with the default parameters.18 Paired-end transcriptome sequencing reads from each sample were aligned with a human reference genome (GRCh38.87) using HISAT v2.0.5 with the default parameters.19 StringTie v1.3.0 was used to assemble and quantify expressed genes and transcripts for each sample and then merge the transcripts from all samples.20 PrepDE was used to compute the raw read counts for each gene. Ballgown v2.6.0 was used to perform multigroup comparisons of gene expression in fragments per kilobase per million.21 Low-variance genes were removed if their expression variance was <1.0 across samples. Significance was defined as a q value <0.05, i.e., a false discovery rate <5%.
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