Library preparation for bulk-sequencing of poly(A)-RNA was done as described previously.115 (link) Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adaptor. Ends of the cDNAs were extended by a template switch oligo (TSO) and full-length cDNA was amplified with primers binding to the TSO-site and the adaptor. NEB UltraII FS kit was used to fragment cDNA. After end repair and A-tailing a TruSeq adapter was ligated and 3’-end-fragments were finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al.,115 (link) the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 63 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2.
Fischer A., Lersch R., de Andrade Krätzig N., Strong A., Friedrich M.J., Weber J., Engleitner T., Öllinger R., Yen H.Y., Kohlhofer U., Gonzalez-Menendez I., Sailer D., Kogan L., Lahnalampi M., Laukkanen S., Kaltenbacher T., Klement C., Rezaei M., Ammon T., Montero J.J., Schneider G., Mayerle J., Heikenwälder M., Schmidt-Supprian M., Quintanilla-Martinez L., Steiger K., Liu P., Cadiñanos J., Vassiliou G.S., Saur D., Lohi O., Heinäniemi M., Conte N., Bradley A., Rad L, & Rad R. (2023). In vivo interrogation of regulatory genomes reveals extensive quasi-insufficiency in cancer evolution. Cell Genomics, 3(3), 100276.
Binding site Bulk Cdna Library Oligo Oligo dt Poly a rna Primers
Corresponding Organization :
Other organizations :
Technical University of Munich, German Cancer Research Center, Wellcome Sanger Institute, Klinikum rechts der Isar, University of Tübingen, University of Eastern Finland, Tampere University, Universitätsmedizin Göttingen, University of Hong Kong, Wellcome/MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge University Hospitals NHS Foundation Trust
Library preparation protocol for bulk-sequencing of poly(A)-RNA
dependent variables
Sequencing results of the cDNA fragments
control variables
Oligo-dT primer containing barcodes, unique molecular identifiers (UMIs), and an adaptor
Template switch oligo (TSO) for cDNA extension
Primers binding to the TSO-site and the adaptor for cDNA amplification
NEB UltraII FS kit for cDNA fragmentation
TruSeq adapter ligation and 3'-end-fragment amplification using primers with Illumina P5 and P7 overhangs
Sequencing on a NextSeq 500 (Illumina) with 63 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2
positive controls
Not explicitly mentioned
negative controls
Not explicitly mentioned
Annotations
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