Direct zol rna prep kit

Tissue and cell samples were dissolved in Isogen (Nippon gene), and total RNAs were prepared by Direct-zol RNA prep kit (Zymo Research). Although other RNA preparation methods are also applicable, we suggest avoiding RNA degradation to capture full-length RNAs. RNA (500 ng) was treated with 25 U of RNA 5′ pyrophosphohydrolase (RppH, New England Biolab) for 1 h at 37 °C in a 50-μl reaction mixture containing 1× Thermopol buffer (New England Biolab). This procedure freed 5′ cap or triphosphate into monophosphate, which can be later ligated by an RNA ligase. The reaction was stopped by phenol-chloroform extraction and ethanol precipitation. Sequencing libraries were constructed by using NEBNext Small RNA Library Prep kit (New England Biolab) according to manufacturer’s instructions. PCR products were run on a 6% native polyacrylamide gel, and a gel region corresponding to 240–380 bp (an insert size of 113–253 bp) was extracted. DNAs were eluted in 300 μl TE, ethanol precipitated, and dissolved in 20 μl of 0.1× TE. Library concentrations were determined by real-time RCR using the KAPA library quantification kit (KAPA Biosystems) on a StepOnePlus (Thermofisher).

All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated. MammoCult media kit, heparin, and hydrocortisone were purchased from Stem Cell Technologies (Vancouver, BC. Canada). F12-K nutrient mix (Kaighn’s) medium was acquired from Gibco (Dublin, Ireland). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Norcross, GA). Collagenase I was purchased from Sigma-Aldrich (St. Louis, MO). Isoliquiritigenin (LigC) and liquiritigenin (LigF) were acquired from ChromaDex (Irvine, CA). 8-prenylapigenin (8-PA) was purchased from Ryan Scientific Inc. (Mount Pleasant, SC). Licochalcone A (LicA), 8-prenylnaringenin (8-PN), and 6-prenylnaringenin (6-PN) were acquired from Sigma-Aldrich (St. Louis, MO) and their purity assessed independently. Xanthohumol (XH) was isolated from hops (Humulus lupulus) as described previously^{51 (link)}. CYP19/MFC high throughput screening kit was purchased from Corning (Corning, NY). Direct-Zol RNA prep kit was acquired from Zymoresearch (Irvine, CA). TRIzol was obtained from Invitrogen (Waltham, MA). PCR reagents, primers, and master mix were purchased from Integrated DNA Technologies (Coralville, IA).

RNA was extracted from 9 hearts of wild type and mvp^−/− fish by using the Direct-zol RNA prep Kit (ZYMO Research, Cat. No. 2052). The libraries were constructed with NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina (NEB, Cat No.6420) and sequenced on illumina Novaseq platform. HISAT2 V2.1.0 was used to map the sample sequencing reads to the GRCz11 reference genome. Gene expression counts were calculated using FeatureCounts v1.6.0. based on current Ensemble annotation. The R package DESeq2 was then employed to make differential gene expression calls. RNAseq data have been deposited in NCBI's Gene Expression Omnibus and can be accessed through GEO Series accession number GSE157170.