rt umi  (New England Biolabs)

Journal: Cell Genomics

Article Title: Integrative high-throughput enhancer surveying and functional verification divulges a YY2-condensed regulatory axis conferring risk for osteoporosis

doi: 10.1016/j.xgen.2024.100501

Figure Lengend Snippet:

Article Snippet: Subsequently, first strand cDNA synthesis was performed using RT-UMI ( ) (NEB, Cat#E6560S, USA).

Techniques: Virus, Plasmid Preparation, Recombinant, Lysis, Gentle, Isolation, RNA HS Assay, Transfection, Luciferase, Reporter Gene Assay, Chromatin Immunoprecipitation, SYBR Green Assay, Functional Assay, Expressing, Sequencing, shRNA, Amplification, Marker, Software

Journal: medRxiv

Article Title: A Massively Parallel Screen of 5′UTR Mutations Identifies Variants Impacting Translation and Protein Production in Neurodevelopmental Disorder Genes

doi: 10.1101/2023.11.02.23297961

Figure Lengend Snippet: A) Cre +/- and Cre -/- offspring from a cross between Ef1a:LSL-eGFP-RPl10 and Vglut1-IRES2-Cre-D were transduced with subpools of the MPRA library (sublibrary 1 in animal 1, sublibrary 2 in animal 2, sublibrary 3 in animal 3, and sublibrary 1 in animal 4). Total RNA extracted from the cortex of each animal at day P21 was subjected to the standard library preparation protocol but varying the reverse transcription (RT) primer used or the addition of Superscript IV reverse transcriptase. After indexing PCR, HSD1000 Tapestation ScreenTapes were used to measure library fragment size distribution and concentration. For all four animals, no final library product was measured without the addition of reverse transcriptase to the cDNA synthesis step. Libraries were generated for all four animals from cDNA primed with the CreOFF_RT_UMI (OFF) primer. Libraries were also generated for all four animals when using the CreON_RT_UMI (ON) primer; however, less product was generated in the final indexing PCR for the Cre -/- animal libraries. B) Sequencing reads from libraries primed with CreOFF_RT_UMI mapped to the reporter construct and barcode sequences at similar rates (93-96%). The CreON_RT_UMI-primed libraries from Cre -/- animal mapped at rates less than 3% (compared to 96% for animal 4) indicating that products amplified from Cre-negative animals were non-specific.

Article Snippet: For fractions collected from HEK cells, RNA was reverse transcribed using the CreOFF_RT_UMI primer (IDT) which appended a 12 bp unique molecular identify (UMI) to each cDNA molecule.

Techniques: Transduction, Concentration Assay, Generated, Sequencing, Construct, Amplification