Backbone-modified nucleic acids are usually more stable enzymatically than their natural counterparts, enabling their broad application as potential diagnostic or therapeutic agents. Moreover, the development of nucleic acids with unnatural backbones has expanded the pool of genetic information carriers and paved the way toward synthetic xenobiology. However, synthesizing these molecules remains very challenging due to the requirement for harsh reaction conditions and the low coupling efficiency during their traditional solid-phase synthesis. Although enzymatic synthesis provides an attractive alternative that also allows the replication and artificial evolution of these molecules, it is crucially dependent on the availability of polymerases capable of synthesizing these backbone-modified nucleotides. Previously, a series of thermostable polymerases that can efficiently synthesize or even amplify backbone-modified DNA or RNA have been evolved through a polymerase evolution method based on phage display. Herein we summarize protocols to use these evolved polymerase mutants to transcribe, reverse transcribe, and PCR amplify backbone-modified nucleic acids. We also outline the polymerase chain transcription method, developed later for the rapid production of RNA or backbone-modified RNA with one of these evolved polymerases, SFM4-3. © 2021 Wiley Periodicals LLC. Basic Protocol 1 Transcription/synthesis of modified DNA/RNA from DNA templates with evolved polymerases SFM4-3 or SFM4-6 Basic Protocol 2 Reverse transcription of modified DNA/RNA with evolved polymerase SFM4-9 Basic Protocol 3 PCR amplification of modified DNA with evolved polymerase SFM4-3 Basic Protocol 4 Polymerase chain transcription for the production of RNA/modified RNA oligonucleotides with evolved polymerase SFM4-3. To help open the clinician dialogue regarding cannabis use in persons with cystic fibrosis (CF) in the United States, we aimed to describe current practices of use assessment and documentation processes related to cannabis. A cross-sectional, anonymous survey study was distributed via email to CF directors and coordinators and to the Cystic Fibrosis Foundation (CFF) listservs of nurse, pharmacist, dietitian, social worker, and psychology care team members. The survey tool included multiple choice, scaled, and open-ended items, which assessed participants' awareness of current cannabis laws in their state, prescribing practices for medical marijuana, screening and documentation practices, knowledge of and what indications participants believe cannabis and cannabidiol (CBD) could be beneficial. Data were analyzed using descriptive statistics. There were 282 survey participants, with majority as providers (28%) and social workers (29%), representing all US regions. Participants varied in terms of frequency of evaluating cannabis use, with 15.4% "always," 48.4% "sometimes," and 41% "rarely," or "never" asking about it. Regarding recreational versus medical cannabis use, 55.4% and 62.5% reported documentation of each type in the medical record, respectively. Participants reported appetite, pain, and nausea as the top three advocated indications for use. About 35% and 72% of participants felt "slightly" or "not at all" prepared to answer patient/family questions about cannabis and CBD, respectively. The approach to cannabis use assessment, documentation, and education across CF care centers is variable. There is a need for care team and patient/caregiver education materials about cannabis/CBD and CF.The approach to cannabis use assessment, documentation, and education across CF care centers is variable. AK 7 There is a need for care team and patient/caregiver education materials about cannabis/CBD and CF.Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs that have been shown to play a role in normal development, homeostasis, and disease, including cancer. CircRNAs are formed through a process called back-splicing, which results in a covalently closed loop with a nonlinear back-spliced junction (BSJ). In general, circRNA BSJs are predicted in RNA sequencing data using one of numerous circRNA detection algorithms. Selected circRNAs are then typically validated using an orthogonal method such as reverse transcription quantitative PCR (RT-qPCR) with circRNA-specific primers. However, linear transcripts originating from endogenous trans-splicing can lead to false-positive signals both in RNA sequencing and in RT-qPCR experiments. Therefore, it is essential to perform the RT-qPCR validation step only after linear RNAs have been degraded using an exonuclease such as ribonuclease R (RNase R). Several RNase R protocols are available for circRNA detection using RNA sequencing or RT-qPCR. These protocols-which vary in enzyme concentration, RNA input amount, incubation times, and cleanup steps-typically lack a detailed validated standard protocol and fail to provide a range of conditions that deliver accurate results. As such, some protocols use RNase R concentrations that are too high, resulting in partial degradation of the target circRNAs. Here, we describe an optimized workflow for circRNA validation, combining RNase R treatment and RT-qPCR. First, we outline the steps for circRNA primer design and qPCR assay validation. Then, we describe RNase R treatment of total RNA and, importantly, a subsequent essential buffer cleanup step. Lastly, we outline the steps to perform the RT-qPCR and discuss the downstream data analyses. © 2021 Wiley Periodicals LLC. Basic Protocol 1 CircRNA primer design and qPCR assay validation Basic Protocol 2 RNase R treatment, cleanup, and RT-qPCR.Protein glycosylation is one of the most common and diverse modifications. Aberrant protein glycosylation has been reported to associate with various diseases. High-throughput and comprehensive characterization of glycoproteins is crucial for structural and functional studies of altered glycosylation in biological, physiological, and pathological processes. In this protocol, we detail a workflow for comprehensive analyses of intact glycopeptides (IGPs), glycosylation sites, and glycans from N-linked glycoproteins. By utilizing liquid handling systems, our workflow could enrich IGPs in a high-throughput manner while reducing sample processing time and human error involved in traditional proteomics sample processing techniques. Together, our workflow enables a high-throughput enrichment of glycans, glycosites, and intact glycopeptides from complex biological or clinical samples. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 Enzymatic digestion of glycoproteins using C4-tips Basic Protocol 2 Intact glycopeptide analysis using C18/MAX-tips Basic Protocol 3 Glycan and glycosite analysis.