Recombinant rnase inhibitor

Total RNA was extracted from each fruit sample using RNAiso Plus (Takara). cDNA was generated in 25-μl reaction mixtures containing 2 μg DNAse I-treated RNA, 200 U M-MLV reverse transcriptase (Takara), 40 U Recombinant RNase Inhibitor (Takara) and 0.1 μΜ oligo (dT) 18 primer. Library construction and quality detection were completed by the Biomarker company (Beijing, China). RNA quality and integrity were analyzed using an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). By using the TruSeq PE Cluster Kit v3-cBot-HS (Illumia), the index-coded samples were clustered following the manufacturer’s instructions. Finally, the libraries were loaded into an Hiseq 2500 (Illumina, USA) for pair-end model sequencing (101 bp).

Whole-cell patch-clamp recordings were performed as previously described (Muñoz-Manchado et al., 2016 (link)) on 5HT3a^EGFP, Pvalb^cre::RCE and Pvalb^cre::tdTomato animals. Prior to brain dissection, animals were transcardially perfused using ice-cold oxygenated cutting solution (in mM): 87 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 26 NaHCO3, 75 sucrose, 12.5 glucose. Patch electrodes (borosilicate glass; resistance 3–8 MΩ; Hilgenberg, GmbH) were filled with 1-2 ul RNase free internal solution containing (in mM): 130 potassium gluconate, 6 NaCl, 10 HEPES, 0.5 EGTA, 4 Na₂-ATP, 0.35 Na₂-GTP, 8 Na₂-phosphocreatine, and 1 U/μl recombinant RNase inhibitor (Takara).

LFS RT-RPA reactions were performed in a 50 μL volume containing 29.5 μL rehydration buffer and 2.5 μL magnesium acetate (280 mM) from the TwistAmp™ nfo kit (TwistDX, Cambridge, UK). Other components included 420 nM each RPA primer (FMDV-LFS-F and FMDV-LFS-R), 120 nM LF probe (FMDV-LFS-P), 200 U MMLV reverse transcriptase (Takara, Dalian, China), 40 U Recombinant RNase Inhibitor (Takara, Dalian, China) and 1 μL of viral RNA or 5 μL of sample RNA. Except for the viral template and magnesium acetate, the other reagents were prepared in a master mix and distributed into a 0.2 mL freeze-dried reaction tube containing a dried enzyme pellet. One μL of viral RNA and 2.5 μL of magnesium acetate were pipetted into the tubes. The RPA was performed in the technician’s closed fist at room temperature for 5, 10, 15 and 20 min as described previously [26 (link), 27 (link)]. The RPA products, which were dual labelled with FAM and Biotin, were detected using LFS as described previously [26 (link), 27 (link)]. A testing sample was considered positive when both the test line and the control line were visible, negative when only the control line was visible, and invalid when the control line was invisible.

Six hundred and thirteen faecal specimens were obtained from outpatients diagnosed with diarrhoea from January 2016 to November 2017, and 30 faecal specimens were obtained from healthy volunteers from Renji Hospital and Children’s Hospital, affiliated with Shanghai Jiaotong University; Tongji Hospital, affiliated with Tongji University; and the Centers for Disease Control in Songjiang district in Shanghai. Patients of all ages with symptoms of acute diarrhoea were considered to be eligible for enrolment. As per the ACG clinical guidelines, acute diarrhoea was defined as the occurrence of defecation 3 or more times per 24 h, with abnormal faecal characteristics, such as loose stool, watery stool, mushy stool, mucosal stool and bloody stool, lasting for less than 14 days [23 (link)]. The exclusion criteria were diarrhoea caused by medicines, poisons, food allergies food intolerance or other diseases. Patients undergoing antibiotic treatments were also excluded. Fresh whole faecal specimens (10 g) were collected in sterilized containers containing 2 mL of normal saline supplemented with recombinant RNase inhibitor (TaKaRa, Japan) to prevent degradation of genetic material from RNA viruses and stored at − 20 °C within 2 h.

For each pull-down sample, 100 μl of streptavidin magnetic beads (S1420S, NEB) were washed with wash/binding buffer (0.5 M NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA) twice, then incubation with probes for 1 h at 4 °C. For biotin-coupled RNA capture, the 5′-end biotinylated PD-L1-lnc probe or control RNA were used. Probes used in these experiments were listed as follows: PD-L1-lnc probe: 5′-3′-CATCCATCATTCTCCCAAGTGAGTCCT; GFP probe: 5′-3′-TGAAGTTCACCTTGATGCCGTT CTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGT. The 0.5 ml cell lysis buffer (Invitrogen) with complete protease inhibitor cocktail (Roche Applied Science, IN, USA) and Recombinant RNase Inhibitor (Takara, Japan) were added into the cell pellets, and lysed by sonication. The cell lysates incubation with RNA-coupled beads followed by centrifugation at 13,000 rpm for 20 min at 4 °C. After rotating for 4 h at 4 °C, the beads were washed with 1 ml lysis buffer (containing 300 mM NaCl) twice, 1 ml low-salt lysis buffer (containing 150 mM NaCl). Half of beads were resuspended in TRIzol™ Reagent for detecting the PD-L1-lnc level, while half of beads were resuspended in RIPA lysis buffer for detecting the c-Myc protein level.