Single-cell transcriptome profiling of tumor-infiltrating CD45+ cells was performed with 10x Genomics Chromium drop-seq platform (V1 chemistry). Freshly sorted CD45+ cells from B16 tumors implanted in Foxp3Cre-YPP or Nrp1L/LFoxp3Cre-YFP mice (female, 5 week of age) on day 14 post inoculation were re-suspended in 1xPBS containing 0.04% BSA. Precisely 2,600 cells were mixed with reverse transcription master mix, gel beads and partition oil were loaded on the GemCode Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell gel-beads in emulsion (GEMs). The reaction mixture/emulsion was removed from the Chromium instrument, and reverse transcription performed, followed by the Dynabead cDNAs clean up and amplification (14 cycles). cDNAs were sheared (Covaris) into ~ 200 bp length. DNA fragment ends were repaired, A-tailed and adaptors ligated. The library was quantified using KAPA Universal Library Quantification Kit (KK4824) and further characterized for cDNA length on a Bioanalyzer using a High Sensitivity DNA kit. Sequencing libraries were loaded at 2.1 pM on an Illumina NextSeq500 with 2 χ 75 paired-end kits using the following read length: 98 bp Read1, 14 bp I7 Index, 8 bp I5 Index and 10 bp Read2.
Universal kapa library quantification kit
Manufactured by Roche
Sourced in Switzerland
The Universal KAPA Library Quantification kit is a tool used for quantifying DNA libraries. It provides a reliable and accurate method to determine the concentration of DNA libraries prior to sequencing.
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Lab products found in correlation
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Miseq system, by Illumina (2 mentions)
C100 thermal cycler, by Bio-Rad (2 mentions)
Hiseq2500 sequencerwas, by Takara Bio (2 mentions)
Smart seq v4 ultra low input rna kit, by Takara Bio (2 mentions)
Nextera xt dna library prep kit, by Illumina (2 mentions)
High sensitivity dna kit, by Agilent Technologies (2 mentions)
T100 thermal cycler, by Bio-Rad (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Gemcode single cell instrument, by 10x Genomics (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Hiseq 2000 sequencer, by Illumina (1 mentions)
Ion chef, by Thermo Fisher Scientific (1 mentions)
Dna 1000 reagents and chips, by Agilent Technologies (1 mentions)
Truseq stranded mrna library prep kit, by Illumina (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
Miseq reagent kit, by Illumina (1 mentions)
Nextseq 500 system, by Illumina (1 mentions)
12 protocols using universal kapa library quantification kit
1
Single-cell transcriptome profiling of tumor-infiltrating immune cells
2
Differential RNA-seq Library Preparation
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Sequencing libraries were prepared from 1 μg aRNA using the NEBNext mRNA LibraryPrep Master Mix Set for Illumina (catalog no. E6110; New England Biolabs, Ipswich, MA). aRNA was fragmented for 2 min instead of the kit-recommended 5 min to account for its shorter length. The rest of the protocol was per manufacturer instructions. Libraries were prepared with indices 4, 6, or 12 from the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1, catalog no. E7335; New England Biolabs). Libraries were quantified using the KAPA Universal Library Quantification Kit (catalog no. KK4824; Kapa Biosystems, Wilmington, MA); three libraries with indices 4, 6, and 12 were multiplexed in equimolar concentration, and 100-bp paired-end sequencing was done on a HiSeq. 2000 sequencer (Illumina, San Diego, CA) using standard Illumina sequencing protocols.
3
Archer VariantPlex Solid Tumor Panel
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The amount of amplifiable DNA (ng) was calculated according to the Archer PreSeq DNA Calculator Assay Protocol (Archer DX, Boulder, CO, USA). After fragmentation of the genomic DNA, libraries were created by the Archer VariantPlex Solid Tumor Kit (Archer DX, Boulder, CO, USA). The solid tumor panel included the following 67 genes: ABL1, AKT1, ALK, APC, ATM, AURKA, BRAF, CCND1, CCNE1, CDH1, CDK4, CDKN2A, CSF1R, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ESR1, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, FOXL2, GNA11, GNAQ, GNAS, H3F3A, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MAP2K1, MDM2, MET, MYC, MYCN, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PIK3R1, PTEN, PTPN11, RB1, RET, RHOA, ROS1, SMAD4, SMARCB1, SMO, SRC, STK11, TERT, TP53, and VHL. The KAPA Universal Library Quantification Kit (Kapa Biosystems, Roche, Basel, Switzerland) was used for the final quantification of the libraries.
4
Archer FusionPlex Expanded Lung Panel
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The Archer FusionPlex Expanded Lung panel (ArcherDX, Invitae, San Francisco, CA) is designed to identify mutations and known and unknown fusions in 17 genes: ALK, BRAF, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, KRAS, MET, NRG1, NTRK1, NTRK2, NTRK3, NUTM1, PIK3CA, RET, and ROS1. For library construction, 250 ng of RNA was used in accordance with the manufacturer’s instructions. Purified libraries were quantified using the KAPA Universal Library Quantification kit (Roche, Basel, Switzerland) and pooled to equimolar concentrations. The Ion 510 & Ion 520 & Ion 530 Kit—Chef and the Ion Chef (ThermoFisher Scientific, Waltham, MA, USA) were used for template preparation and chip loading. Sequencing was performed using the S5 Gene Studio instrument (ThermoFisher Scientific, Waltham, MA, USA). Results were analyzed using the Archer Suite Analysis software version 6.2.7 (ArcherDx, Invitae, San Francisco, CA). A sample was considered contributory when the following quality control criteria were obtained: Presequencing Cp value <28.5, a minimum of 500 000 reads (total fragments) and a minimum of 95% of reads (unique fragments) on target. A fusion was considered as present when a minimum of 3 single start sites were obtained, a minimum of 10 reads supported the fusion and the fusion was in frame.
5
Whole transcriptome RNA-seq library preparation
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Whole mRNA transcriptome library preparation and sequencing was performed using methods as previously described35 (link). Briefly, RNA sequencing libraries were prepared with TruSeq Stranded mRNA Library Prep Kit (Illumina) using 500 ng – 1 µg RNA. Quality of libraries were validated on an Agilent Bioanalyzer using DNA 1000 reagents and chips (Agilent Genomics, Waldbronn, Germany) to quantify library sizes and confirm the absence of primer dimers. Libraries were quantified using a KAPA Universal Library Quantification kit (Roche Diagnostics Limited) on a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) and library concentrations were adjusted for library size. Pooled libraries of 12–15 pM concentrations were sequenced on a MiSeq System (Illumina) using a MiSeq Reagent Kit v3 (Illumina) with 2 × 75 cycles.
6
Kapa Hyper Prep Kit for ChIP-Seq
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Libraries were made with the Kapa Hyper Prep kit (Roche, KK8502), starting with 2.5 ng of IP DNA, ad amplified by 15 cycles of PCR amplification, according to the manufacturer’s protocol. Libraries were quantified and sized by running them on an Agilent Tapestation, measuring concentration of QPCR (Kapa Universal Library Quantification kit, Roche, KK4824). The libraries were run on an Illumina 2500, v4 chemistry, using a single read 50 protocol. ChIP-Seq analysis is described in Supplementary Methods .
7
3'-RACE-seq Library Quantification and Sequencing
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3′-RACE-seq library concentrations were measured using the KAPA Library Quantification Universal Kit (Kapa Biosystems). Sequencing was performed using an Illumina MiSeq System and MiSeq Reagent Kit (version 3, 2 × 75 bp read length) by applying standard paired-end run procedures.
8
Quantifying and Sequencing 3'-RACE Libraries
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Concentration of the 3′-RACE-seq libraries was measured with the KAPA Library Quantification Universal Kit (Kapa Biosystems). Sequencing was performed using an Illumina NextSeq 500 System and NextSeq 500 v2 High Output Kit (2 × 75 cycles), applying standard procedures of a pair-end run.
9
RNA-seq Analysis Pipeline for Murine Samples
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RNA extraction was performed using the TRIzol Reagent according to the manufacturer’s instructions (15596018, ThermoFisher).
RNA libraries were prepared using TruSeq Stranded mRNA kit (Illumina). The concentration of indexed libraries was quantified by RT–qPCR using the Universal Kapa Library Quantification Kit (KAPA Biosystems). Final libraries were normalised and sequenced on an Illumina HiSeq 2000 sequencer.
Raw fastq-files were aligned against the murine genome version mm10 using TopHat (v2.0.13) with all default options. BAM files containing the alignment results were sorted according to the mapping position. mRNA quantification was performed using FeatureCounts from the Subread package against the GRCm38-gencode transcripts database version seven (gencode.vM7.annotation.gtf) and the GRCh38-genocode transcripts database version 24 (gencode.v24.annotation.gtf) to obtain read counts for each individual Ensembl gene. The read count table of the dataset was normalised separately using DESeq2.
RNA libraries were prepared using TruSeq Stranded mRNA kit (Illumina). The concentration of indexed libraries was quantified by RT–qPCR using the Universal Kapa Library Quantification Kit (KAPA Biosystems). Final libraries were normalised and sequenced on an Illumina HiSeq 2000 sequencer.
Raw fastq-files were aligned against the murine genome version mm10 using TopHat (v2.0.13) with all default options. BAM files containing the alignment results were sorted according to the mapping position. mRNA quantification was performed using FeatureCounts from the Subread package against the GRCm38-gencode transcripts database version seven (gencode.vM7.annotation.gtf) and the GRCh38-genocode transcripts database version 24 (gencode.v24.annotation.gtf) to obtain read counts for each individual Ensembl gene. The read count table of the dataset was normalised separately using DESeq2.
10
Illumina HiSeq2500 RNA Sequencing
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Library preparation for sequencing on an Illumina HiSeq2500 sequencer
was carried out using the SMART-seq v4 Ultra low Input RNA kit (Clontech)
following the manufacturer’s instructions. All steps were carried out on
ice unless otherwise specified. Reverse transcription, PCR cycles and incubation
steps utilised a BioRad T100 Thermal Cycler. Amplification of cDNA by LC PCR
used a 10-cycle protocol. After bead purification, cDNA library concentration
was measured (High Sensitivity DNA kit, Agilent Technologies).
Sequencing libraries were generated using the Nextera XT DNA Library
Prep Kit (Illumina) using 150 pg cDNA as input following the
manufacturer’s instructions with the following modification. Following
library amplification and bead purification the final fragment size was analysed
and libraries quantified using the Universal KAPA Library Quantification kit
(Kapa Biosystems) and a Bio-Rad C100 thermal cycler. An equal amount of cDNA was
used to pool up to four samples, which were sequenced in one lane. Sequencing
was carried out to a depth of 50 million 100 bp paired-end reads per
library.
was carried out using the SMART-seq v4 Ultra low Input RNA kit (Clontech)
following the manufacturer’s instructions. All steps were carried out on
ice unless otherwise specified. Reverse transcription, PCR cycles and incubation
steps utilised a BioRad T100 Thermal Cycler. Amplification of cDNA by LC PCR
used a 10-cycle protocol. After bead purification, cDNA library concentration
was measured (High Sensitivity DNA kit, Agilent Technologies).
Sequencing libraries were generated using the Nextera XT DNA Library
Prep Kit (Illumina) using 150 pg cDNA as input following the
manufacturer’s instructions with the following modification. Following
library amplification and bead purification the final fragment size was analysed
and libraries quantified using the Universal KAPA Library Quantification kit
(Kapa Biosystems) and a Bio-Rad C100 thermal cycler. An equal amount of cDNA was
used to pool up to four samples, which were sequenced in one lane. Sequencing
was carried out to a depth of 50 million 100 bp paired-end reads per
library.
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