SORT-seq Partially Automated scRNA-seq

scRNA-seq libraries were generated using the SORT-seq protocol (54 (link)), a partially robotized method based on the CEL-Seq2 protocol (55 (link)). Briefly, single cells were lysed at 65°C for 5 min, and second-strand mixers and reverse transcriptase were then added to the wells using the Nanodrop II liquid handling platform (GC Biotech). After reverse-transcribing the mRNA of each cell, double-stranded complementary DNAs (cDNAs) from single cells were pooled, and in vitro transcription was performed for linear amplification, which resulted in amplified RNA. TruSeq small RNA primers (Illumina) were used to prepare the Illumina sequencing libraries, and these DNA libraries were then sequenced paired-end at 75–base pair read length using the Illumina NextSeq (performed commercially by Single Cell Discoveries, Utrecht, The Netherlands). Both the RNA yield of the amplified RNA and the quality and concentration of the final cDNA libraries were assessed by Bioanalyzer (Agilent).

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Souilhol C., Tardajos Ayllon B., Li X., Diagbouga M.R., Zhou Z., Canham L., Roddie H., Pirri D., Chambers E.V., Dunning M.J., Ariaans M., Li J., Fang Y., Jørgensen H.F., Simons M., Krams R., Waltenberger J., Fragiadaki M., Ridger V., De Val S., Francis S.E., Chico T.J., Serbanovic-Canic J, & Evans P.C. (2022). JAG1-NOTCH4 mechanosensing drives atherosclerosis. Science Advances, 8(35), eabo7958.

Publication 2022

TruSeq small RNA primers NextSeq Bioanalyzer

Base pair Cdna libraries Cdnas Cell Dna libraries Double stranded dnas Mrna Reverse transcriptase Rna primers Scrna seq Transcription

Corresponding Organization : Insigneo

Other organizations : Southwest Medical University, University of Chicago, Addenbrooke's Hospital, University of Cambridge, Queen Mary University of London, University of Münster, Hirslanden Klinik Im Park, University of Oxford, Ludwig Cancer Research

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Variable analysis

independent variables

The SORT-seq protocol used to generate scRNA-seq libraries

dependent variables

RNA yield of the amplified RNA
Quality and concentration of the final cDNA libraries

control variables

Single cells were lysed at 65°C for 5 min
Second-strand mixers and reverse transcriptase were added to the wells using the Nanodrop II liquid handling platform (GC Biotech)
Double-stranded complementary DNAs (cDNAs) from single cells were pooled and subjected to in vitro transcription for linear amplification
TruSeq small RNA primers (Illumina) were used to prepare the Illumina sequencing libraries
DNA libraries were sequenced paired-end at 75–base pair read length using the Illumina NextSeq

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