Journal: bioRxiv
Article Title: SCIMETAR-seq tracks immunophenotype, demethylation, mutations, and transcriptomes in single cells undergoing HMA therapy
doi: 10.64898/2026.04.26.720516
Figure Lengend Snippet: (a) Details of L1 ES-qPCR. (i) L1 5’UTR ES-qPCR oligonucleotides, (ii) L1 reference oligonucleotides. (iii) Sequence of L1 ES-qPCR reactions. (1) Restriction digestion; (2) Annealing of L1-specific foligos; (3) extension of 3’-ends created by restriction digestion – if the 3’-ends are annealed to a foligo; (4) copying of (green) foligo-tagged templates or (red) reference templates by PCR; (5) detection of products by quenched probes. (b) Sensitivity of (solid symbols) original versus (open symbols) updated L1 ES-qPCR reagents. (c) Dependence of L1 ES-qPCR FAM signals on HpaII- digestion. (d) Ct plotted as function of log 2 [DNA]); used for (e) PCR efficiency calculations (top) and relative L1 demethylation calculations (bottom). (f) Examples of targeted amplicon recoveries from (i) Hpa II-digested genomic gDNA or (ii) Hpa II-digested first strand cDNA. (g) Averaged heat map of gene coverage by the pilot SCIMETAR-seq. Low recovery in the first 2 rows was due to evaporation during primary PCR. (h) Rationale for filtering of L1 ES-qPCR outliers: All single cells contain the same number of L1 refernce DNA templates, but will vary in L1 methylation, Therefore, reference (HEX) Ct that fall outside 2xMAD were excluded as failed outliers (white dots). Note that not all HEX Ct outliers are also FAM (demethylation) Ct outliers. (i) Detail of complete target amplicons: (green) Hpa II sites, (red) target mutations sites, (purple N) I5 or i7 index, (X…X) well barcodes, ( underlined ) exonic sequence.
Article Snippet: Reaction mixtures contained 20 mM Tris-HCl (pH ∼8.3; Merck, 648310-M), 50 mM KCl (Merck, 60142-100mL-F), 7 mM MgCl2 (Merck, M1028-10X1mL), 200 μM dNTP mix (Thermo Fisher Scientific, R0181), 0.5 U of Hot Start Taq DNA polymerase (New England Biolabs, M0495L), and 2 U of HpaII (New England Biolabs, R0171L).
Techniques: Sequencing, Amplification, Evaporation, Methylation
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![(a) Details of L1 ES-qPCR. (i) L1 5’UTR ES-qPCR oligonucleotides, (ii) L1 reference oligonucleotides. (iii) Sequence of L1 ES-qPCR reactions. (1) Restriction digestion; (2) Annealing of L1-specific foligos; (3) extension of 3’-ends created by restriction digestion – if the 3’-ends are annealed to a foligo; (4) copying of (green) foligo-tagged templates or (red) reference templates by PCR; (5) detection of products by quenched probes. (b) Sensitivity of (solid symbols) original versus (open symbols) updated L1 ES-qPCR reagents. (c) Dependence of L1 ES-qPCR FAM signals on <t>HpaII-</t> digestion. (d) Ct plotted as function of log <t>2</t> <t>[DNA]);</t> used for (e) PCR efficiency calculations (top) and relative L1 demethylation calculations (bottom). (f) Examples of targeted amplicon recoveries from (i) Hpa II-digested genomic gDNA or (ii) Hpa II-digested first strand cDNA. (g) Averaged heat map of gene coverage by the pilot SCIMETAR-seq. Low recovery in the first 2 rows was due to evaporation during primary PCR. (h) Rationale for filtering of L1 ES-qPCR outliers: All single cells contain the same number of L1 refernce DNA templates, but will vary in L1 methylation, Therefore, reference (HEX) Ct that fall outside 2xMAD were excluded as failed outliers (white dots). Note that not all HEX Ct outliers are also FAM (demethylation) Ct outliers. (i) Detail of complete target amplicons: (green) Hpa II sites, (red) target mutations sites, (purple N) I5 or i7 index, (X…X) well barcodes, ( underlined ) exonic sequence.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_16/10__64898_slash_2026__04__26__720516/10__64898_slash_2026__04__26__720516___F4.large.jpg)