Ultra 2 fs kit

Half of the pre-amplified cDNA produced by ScNaUMI-seq was processed into an Illumina sequencing library using the NEB Ultra II FS kit (NEB cat. no. E7805S). Libraries were paired-end sequenced on a NovaSeq 4000 instrument. The other half was further amplified according to the ScNaUMI-seq protocol, ONT sequencing libraries were prepared using the Ligation Sequencing Kit (ONT SQK-LSK110) and sequenced using three MinION R9.4.1 flow cells.

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.

Library preparation for bulk-sequencing of poly(A)-RNA was done as described previously^{78 (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 adapter. 5’-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 adapter. 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.^{78 (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 59 cycles for the cDNA in read1 and 18 cycles for the barcodes and UMIs in read2. Data was processed using the published Drop-seq pipeline (v1.0) to generate sample- and gene-wise UMI tables^{79 (link)}. Reference genome (GRCm38) was used for alignment. Transcript and gene definitions were used according to the GENCODE version M25. Differential expression analysis was performed using R and DESeq2 (1.38.0). A P value of <0.05 was used to determine differentially expressed genes. The data

A sequencing library was prepared from genomic DNA by using a NEB Ultra II FS kit with dual-indexed barcoding and sequenced on an Illumina MiSeq, yielding a total of 901,246 single-end 150bp reads (>895X coverage). Quality control checks using FastQC v.0.11.7 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc; last accessed 2020/04/30) indicated that the data were of high quality (i.e., no adapters were present and base quality scores were >30, equivalent to an error rate of <0.1%; Figure S1). Consequently, no adapter contaminations were trimmed off of the 3′-end of the reads by scythe v.0.991 (a Naive Bayesian approach to detect and remove contamination) and sickle v.1.33 (a tool for quality-based read trimming) flagged only ∼0.1% of the reads for containing base qualities <20. With no significant changes to our dataset, additional read processing prior to assembly was thus deemed unnecessary. Following Russell (2018) (link), reads were de novo assembled using Newbler v2.9, resulting in a single linear contig of size 150,935bp, which was checked for completeness, accuracy, and phage genomic termini using Consed v.29 (Gordon et al. 1998 (link)). All software was executed using default settings.