ont native barcoding kit 24 v14  (New England Biolabs)

Journal: bioRxiv

Article Title: EXTRA-seq: a genome-integrated extended massively parallel reporter assay to quantify enhancer-promoter communication

doi: 10.1101/2024.12.08.627402

Figure Lengend Snippet: a Overview of the EXTRA-seq approach. Middle: A lox-based landing pad is integrated at a focal location, replacing part of the 5’ UTR, the TSS, and the TSS-upstream regulatory region of interest (middle, top). The landing pad is replaced with a library encoding different enhancer (E) and promoter (P) modifications with unique barcodes (BCs) in the UTR. mRNA and gDNA are amplified using a fixed library (lib) and either exon-(mRNA) or intron-(gDNA) specific primers. The log fold change (logFC) of mRNA over gDNA ratios reflects construct-level expression. Left : Strategy to synthesize large, barcoded libraries with simultaneous modification to enhancers and promoters, and to map modifications to each BC. Right : Experimental strategy illustrating the use of BCs to capture pooled genotypes. b Validating mapping accuracy using a plasmid library containing pre-determined, enhancer-centric transcription factor binding site (TFBS)-to-BC associations. BCs contain a fixed TFBS-specific (5bps) and a random (8bps) part. Number of unique 8mers per fixed 5mer is shown across TFBSs. c Description of the rs143348853 variant locus at the AXIN2 gene. A 5bp deletion creates a new MEF2 TFBS in the alternative (ALT) genotype enhancing AXIN2 expression. d AXIN2 expression levels (FPKM = fragments per kilobase of transcript per million mapped reads) across individually derived LCL cell lines from human donors split by their genotype for rs143348853. Homozygous reference (REF; no deletion) and heterozygous (REF/ALT) LCLs are shown to illustrate expected expression changes due to one copy of the ALT genotype. e Illustration of the AXIN2 locus after placement of the EXTRA-seq cassette. f The fractions of ‘expressed’ BCs per replicate (non-zero counts in the mRNA library) are shown for either the REF or ALT EXTRA-seq allele (Mann Whitney test; p-value = 0.029). g Raw EXTRA-seq activities (averaged across replicates) per BC grouped by genotype (Mann Whitney test; p-value = 9.1*10 − 9 ). Boxplots show the mean and quartiles with whiskers extending to 1.5 times the interquartile range. h Summarized log fold change (logFC) in expression using the mpralm function , . Whiskers indicate the scaled standard deviations associated with each fit.

Article Snippet: Library plasmids were digested with restriction enzymes (e.g. AgeI and SalI or BamHI when integrated within the STARR-seq vector) and the resulting DNA was gel purified before ligating library-specific Oxford Nanopore Technologies (ONT) barcodes (SQK-NBD114.24) and adaptor sequences for ONT sequencing.

Techniques: Amplification, Construct, Expressing, Modification, Plasmid Preparation, Binding Assay, Variant Assay, Derivative Assay, MANN-WHITNEY