Rnase inhibitor

The method for the isolation of HCC-LM3 was provided previously [15 (link)]. In short, the cells were treated with cycloheximide (CHX) for 5 min. Then polysome lysis buffer were applied to cells and incubated for 30 min. After centrifugation, the supernatant was loaded onto continuous 15–50% sucrose gradients buffer containing 50 U/ml RNase inhibitor (Solarbio, China). The cells fractions were collected using Brandel Fractionation System (USA). Extraction and total RNA of each fraction was measured by RT-PCR, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a positive control.

Tris buffer solution (1 mol L⁻¹, pH 8.0), dNTP mixture (25 mmol L⁻¹) and RNase inhibitor were purchased from Solarbio Life Sciences (China). Sodium chloride and magnesium chloride were purchased from Macklin Biochemical Co., Ltd (Shanghai, China). The microRNA, padlock probe and three oligonucleotides (Table 1) were synthesized and HPLC-purified by Sanggon Biotech Co., Ltd. (Shanghai, China). Ultrapure water was purchased Dongsheng Biotech Co., Ltd. (Guangzhou, China). T4 DNA ligase, phi29 polymerase, exonuclease I (EXO I) and bovine serum albumin (BSA) were purchased from New England Biolabs (Ipswich, MA, USA).

Nuclei isolation from adipose tissue was performed according to a published protocol with slight modification (Van Hauwaert et al., 2021 (link)). Briefly, adipose tissues were minced in a Petri dish containing 500 mL nuclei isolation buffer (NIB) that contains 250 mM sucrose (Solarbio, Cat# S8271), 10 mM HEPES (Solarbio, Cat# H8090), 1.5 mM MgCl₂ (Sigma-Aldrich, Cat# M8266), 10 mM KCl (Sigma-Aldrich, Cat# P5405), 0.001% IGEPAL CA-630 (NP-40) (Sigma-Aldrich, Cat# I3021), 0.2 mM DTT (Sigma-Aldrich, Cat# D9779), and 1 U/μL RNase inhibitor (Solarbio, Cat# R8061) in DEPC-treated water. The samples were further homogenized using a 2 mL Dounce homogenizer (Sigma-Aldrich, Cat# D8938), applying three strokes with the loose pestle. The homogenate was filtered through a 70 μm cell strainer (Falcon, Cat# 352350) and centrifuged at 500 × g for 5 min at 4°C. The nuclei pellet was resuspended in 2 mL NIB and centrifuged at 300 × g for 3 min at 4°C. Finally, the nuclear pellet was resuspended in 300 μL nuclei resuspension buffer that contains 2% BSA (Sigma-Aldrich, Cat# A1933), 1.5 mM MgCl₂, and 1 U/μL RNase inhibitor in PBS. All solutions were sterile filtered prior to use. All procedures were performed on ice.