Nevena Milivojević1*, Uršula Prosenc Zmrzljak2, Biljana Ljujić3, Valentina Đorđević4, Marina Gazdić Janković3, Marko Živanović1,5, Feđa Puač6, Miloš Ivanović7, and Nenad Filipović5,8
1Institute for Information Technologies, University of Kragujevac, Jovana Cvijića bb, 34000 Kragujevac, Serbia
2BIA Separations CRO Laboratory, Teslova ulica 30, 1000 Ljubljana, Slovenia
3Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovića 69, 34000 Kragujevac, Serbia
4Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11042 Belgrade, Serbia
5BioIRC – Bioengineering Research and Development Center, University of Kragujevac, Prvoslava Stojanovića 6, 34000 Kragujevac, Serbia
6Labena d.o.o. Serbia, Bulevar Zorana Đinđića 123G, 11070 Belgrade, Serbia
7Faculty of Science, University of Kragujevac, Radoja Domanovića 12, 34000 Kragujevac, Serbia
8Faculty of Engineering, University of Kragujevac, Sestre Janjić 6, 34000 Kragujevac, Serbia
nevena_milivojevic [at] live.com
Abstract
Whole 3` transcriptome profiling at the single cell level opens up new abilities for researchers to answer complex questions. Thousands of individual cells per sample are Barcoded separately to index the transcriptome of each cell individually. It is done by partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where cells are delivered at a limiting dilution, such that the majority (~90-99%) of generated GEMs contain no cell. The 16 bp 10x Barcode and 12 bp UMI are encoded in Read 1, while the poly(dT) primers are used in this protocol for generating Single Cell 3ʹ Gene Expression libraries. After GEM generation, copartitioned cells are lysed and reverse transcription (RT) was performed after which all cDNA from single cell share a common Barcode. Full-length cDNA was amplified via PCR to generate sufficient mass for library construction. This is followed by enzymatic fragmentation and size selection to optimize the cDNA amplicon size. Library construction was finished via End Repair, A-tailing, Adaptor Ligation, and PCR. P5, P7, i7 and i5 sample index, and TruSeq Read 2 (read 2 primer sequence) were added. TruSeq Read 1 and TruSeq Read 2 are standard Illumina sequencing primer sites used in paired-end sequencing. The library prepared in this way, containing the P5 and P7 primers, is ready for Illumina amplification.
Keywords: single-cell analysis, mRNA, bioinformatics, transcriptome, sequencing
Acknowledgement: This research is funded by Labena Slovenia 10xGenomics Grant Challenge (Project title: Deciphering the effects of nanosized polystyrene particles using lab-on-chip technology and transcriptome profile). This research was supported by Labena Serbia, the Ministry of Science, Technological Development and Innovation of the Republic of Serbia, contract number 451-03-47/2023-01/200378 (Institute for Information Technologies Kragujevac, University of Kragujevac), 451-03-47/2023-01/200111 (Faculty of Medical Sciences, University of Kragujevac), as well as Junior projects of Faculty of Medical Sciences, University of Kragujevac JP 25/19, JP 05/20, JP 06/20 and JP 24/20. This work is supported by the European Union’s Horizon 2020 research and innovation program under grant agreement No 952603 (SGABU). This article reflects only the author’s view. The Commission is not responsible for any use that may be made of the information it contains.
