Phenol chloroform

Deparaffinization of 40 µm tissue sections was performed with 100% xylene (Sigma Aldrich; Merck KGaA, Darmstadt, Germany) followed by 100% ethanol as described previously (16 (link)). Deparaffinized samples were suspended in proteinase K-contained buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Following extraction using phenol-chloroform (Sigma Aldrich; Merck KGaA), DNA samples were treated with ethanol (Sigma Aldrich; Merck KGaA) precipitation and resuspended in deionized water. cfDNA blood collection tubes (Streck, Inc., Omaha, NE, USA) were used for the collection of 10 ml blood samples. Procurement of 1.2 ml of plasma from every patient was performed prior to cfDNA extraction using centrifugation twice (4°C; 1,600 × g for 10 min). cfDNA was isolated with the QIAamp Circulating Nucleic Acid kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. Validation of adopted protocols for the present study was obtained from previous findings (17 (link)). A total of 2 ml of total peripheral blood was used to process WBC DNA as a control, using a FlexiGene DNA kit (Qiagen, Inc.). Quantification of isolated DNA samples was performed using a NanoDrop spectrophotometer (Thermo-Fisher Scientific, Inc.), and by fluorimetry, which was conducted using Qubit dsDNA high-sensitivity and/or broad-range assay kits (Thermo Fisher Scientific, Inc.).

To assess and quantify total DNA content, 30 mg of
lyophilized native and decellularized lung tissues (n=3)
were treated with digesting buffer solution containing 50
mM Tris-HCl (pH=8, 1185-53-1, Sigma-Aldrich, USA),
50 mM EDTA (60-00-4, Sigma-Aldrich, USA), 10 mM
NaCl, and 1% SDS in the presence of 10 µl proteinase K
(25530015, Invitrogen, USA) overnight at 65°C in water
bath. Samples were purified using phenol-chloroform
(136112-00-0, Sigma-Aldrich, USA, 500 µl to each sample)
extraction. DNA was precipitated from the upper aqueous
layer with 100% ethanol. The extracts were afterwards
washed with 70% ethanol (64-17-5, Merck, USA) and
dried under laminar hood. After dissolving the resultant
pellet in 50 µl DDW, absorbance was measured at 260 nm
using a spectrophotometer (Multiskan spectrum, Thermo
scientific, Germany) to determine the concentration of
DNA.

Mouse strains were maintained in the same genetic background (C57BL/6J). Genotyping was performed by conventional Polymerase Chain Reaction (PCR) on genomic DNA isolated from mouse tails using standard procedures. Briefly, mouse tails were dissolved in Tail buffer (100 mM Tris–HCl pH 8.0, 200 mM NaCl, 5 mM EDTA, and 0.2% SDS) with 50 μg/ml Proteinase K (Sigma) overnight at 55°C. Following a protein extraction step with Phenol/Chloroform (Sigma), genomic DNA was precipitated from the aqueous phase with 100% ethanol and finally resuspended in TE buffer (10 mM Tris–HCl pH 8.0 and 1 mM EDTA). Genotyping procedures were as described for Aldh1b1 alleles (http://www.velocigene.com/komp/detail/11807)). The Aldh1b1^tm1lacZ knockin mouse strain was generated as previously described 21. Animal maintenance and experimentation were conducted in accordance with the FELASA recommendations and the ethical and practical guidelines for the care and use of laboratory animals set by the competent veterinary authorities in the authors' institutions.

For RNase R treatment, total RNA was incubated with or without RNase R (3 U/mg, Epicentre, Shanghai, China) at 37°C for 15 min, followed by the purification with phenol-chloroform (Sigma-Aldrich, St. Louis, MO, USA). And then, RT-qPCR assay was performed to detect the levels of circNHSL1 and NHSL1.
For subcellular fractionation assay, HGC-27 and AGS cells were suspended and centrifuged in cytoplasm lysis buffer. After collecting the cytoplasmic supernatant, the rest was incubated and centrifuged in nucleus lysis buffer. Whereafter, TRIzol reagent (TaKaRa) was used for the extraction of RNAs from cytoplasmic and nuclear. At last, the levels of circNHSL1, U6 (nucleus control), and GAPDH (cytoplasm control) were measured by RT-qPCR assay in cytoplasmic and nuclear.