5P expression and TCR libraries were clustered on an Illumina NovaSeq pair-end read flow cell and sequenced for 28 cycles on R1 (10x barcode and the UMIs), followed by 8 cycles of I7 Index (sample Index), and 91 bases on R2 (transcript), obtaining about 250 million clusters for 5P expression and 50 million for TCR libraries. Primary processing of sequencing images was done using Illumina’s Real Time Analysis software (RTA). 10x Genomics Cell Ranger Single Cell Software suite (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger was used to perform sample ) was used for demultiplexing, alignment (hg19), filtering, UMI counting, single-cell 5′ end gene counting, TCR assembly, annotation of paired VDJ and performing quality control using the manufacturer’s parameters.
Real time analysis software rta
Manufactured by Illumina
The Real Time Analysis (RTA) software is a core component of Illumina's sequencing instruments. It is responsible for processing raw data generated during the sequencing run and generating usable sequence information. The RTA software performs real-time image analysis, base calling, and other necessary data processing steps to produce the final sequencing output.
Automatically generated - may contain errors
2 protocols using real time analysis software rta
1
Single-Cell Transcriptome and TCR Profiling
2
Exome-enriched RNA sequencing protocol
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Briefly, stranded RNAseq libraries were generated as per the manufacturer's recommendations, but without the transcriptome enrichment step (pre-capture libraries).
Transcriptome enrichment was achieved by the hybridization of the pre-capture library to the exome panels tested. Since the probe baits were biotinylated, hybridized libraries were captured using streptavidin beads (ThermoFisher, Waltham, MA) and PCR amplified-on-beads to generate a post-capture library. All post-capture libraries were subjected to quality control on an Agilent Bioanalyzer and normalized to 2nM. The postcapture libraries obtained from each capture platform were pooled, and each pool sequenced on one lane of a paired-end read flow cell for 2x100 cycles on a HiSeq4000 to obtain ~40M reads per sample. The primary processing of sequencing images was done using Illumina's Real Time Analysis software (RTA). CASAVA 1.8.2 software was then used to demultiplex samples and generate raw reads and respective quality scores (Supplementary Tables 1 and2). Libraries were made as described above using the NEBNext Ultra II Directional kit (PN-E7760, New England Biolabs, Ipswich, MA). 5µg of pooled indexed libraries (500ng each)
Transcriptome enrichment was achieved by the hybridization of the pre-capture library to the exome panels tested. Since the probe baits were biotinylated, hybridized libraries were captured using streptavidin beads (ThermoFisher, Waltham, MA) and PCR amplified-on-beads to generate a post-capture library. All post-capture libraries were subjected to quality control on an Agilent Bioanalyzer and normalized to 2nM. The postcapture libraries obtained from each capture platform were pooled, and each pool sequenced on one lane of a paired-end read flow cell for 2x100 cycles on a HiSeq4000 to obtain ~40M reads per sample. The primary processing of sequencing images was done using Illumina's Real Time Analysis software (RTA). CASAVA 1.8.2 software was then used to demultiplex samples and generate raw reads and respective quality scores (Supplementary Tables 1 and2). Libraries were made as described above using the NEBNext Ultra II Directional kit (PN-E7760, New England Biolabs, Ipswich, MA). 5µg of pooled indexed libraries (500ng each)
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