High sensitivity dna bioanalyzer

After FACS sorting, cells were processed using the Chromium Single Cell Gene Expression 3′ Library and Gel Bead kit following the manufacturer’s instructions (10x Genomics). Cells were counted and checked for viability using a Vi-CELL XR cell counter (Beckman Coulter) and then injected into microfluidic chips to form Gel Beads-in-Emulsion (GEMs) in the 10x Chromium instrument. Reverse transcription was performed on the GEMs, and reverse-transcribed products were purified and amplified. Gene expression libraries and hashtag libraries were made from the cDNA, profiled using a Bioanalyzer High Sensitivity DNA kit (Agilent Technologies), and quantified with a Kapa Library Quantification kit (Kapa Biosystems). HiSeq 4000 (Illumina) was used to sequence the libraries.
For TCR-sequencing, the cells were processed using the Chromium Single Cell Immune Profiling 5’ v2 kit (10x Genomics). After reverse transcription and cDNA amplification, TCR enrichment was performed using mouse T cell primer mixes according to 10x’s recommendations. Subsequently, libraries were constructed for gene expression, TCR, and hashtags. Libraries were profiled with the Bioanalyzer High Sensitivity DNA (Agilent Technologies) and quantified with a Kapa Library Quantification kit (Kapa Biosystems). HiSeq 4000 (Illumina) was used to sequence the libraries.

Lymphocytes were isolated from BM samples by density-gradient centrifugation (ficoll^® Paque Plus; Sigma-Aldrich). Total RNA was extracted from 10⁸ cells with an RNeasy midi kit following the manufacturer’s protocol (Qiagen) and enriched for mRNA using paramagnetic separation (μMACS mRNA Isolation kit; Miltenyi Biotec, Leiden, Netherlands). cDNA was prepared from 300 ng of mRNA using dT₁₈ primers and Superscript III reverse transcriptase (Thermo Fisher Scientific) at 50°C for 80 min. Recombined IgH fragments were subsequently amplified by multiplex PCR using primers for human IgHV region and rat Cγ region with Q5 Hot Start High Fidelity polymerase (NEB, Ipswich, MA, USA) as described previously (22 (link)). Amplicons were size selected on a 2% agarose gel and quantified. Quality was checked with a Bioanalyzer (High Sensitivity DNA, Agilent Technologies, Diegem, Belgium). Four randomly selected libraries were pooled in equimolar concentrations and sequenced on a 318™ Chip v2 (Thermo Fisher Scientific) using multiplex identifiers (MIDs) with the Ion OneTouch™ Template OT2 400 Kit and the Ion PGM Sequencing 400 Kit (Thermo Fisher Scientific) on the Ion Torrent Ion Personal Genome Machine (PGM™) System (Thermo Fischer Scientific).

The average base-pair size for each library was determined using an Agilent High Sensitivity DNA Bioanalyzer or an Advanced Analytical Fragment Analyzer, and final library concentration was determined using a Qubit High Sensitivity DNA assay kit (Invitrogen; Q32854). All libraries were sequenced on a NextSeq 500. Pharynx atlas single-cell RNA-seq libraries were sequenced using a 75 cycle NextSeq 500/550 High Output Kit v2 (Illumina; FC-404-2005) at 26(Read 1)/50(Read 2). All single-cell RNA-seq libraries pertaining to the Foxn1^nu mice were sequenced using a 75 cycle NextSeq 500/550 High Output Kit v2.5 (Illumina; 20024906) at 26(Read 1)/50(Read 2). Single-cell ATAC-seq libraries were sequenced using a 150 cycle NextSeq 500/550 High Output Kit v2.5 (Illumina; 20024907) at 65(Read 1)/65(Read 2). In addition, libraries were indexed for multiplexed sequencing.

RNA-seq libraries of 4-TU labeled samples were prepared using the ScriptSeq V2 RNA-Seq Library Preparation Kit (cat. SSV21124, Illumina, San Diego, CA) following the manufacturer’s protocol. In brief, cDNA was synthesized using ScriptSeq cDNA synthesis primer, StarScript AMV reverse transcriptase, and 100 mM DTT. The 3′ end was tagged using Terminal tagging premix and DNA polymerase. cDNA was purified using 1.8× AMPure XP, and libraries were PCR amplified by adding Illumina adapter sequence and an index in the library. Final libraries were again purified with 1× Agencourt AMPure XP (cat. A63881, Beckman Coulter, Brea, CA) and libraries were validated using Qubit 2.0 (Thermo Fisher, Waltham, MA) and High sensitivity DNA bioanalyzer (Agilent, Santa Clara, CA). Libraries were diluted to 2 nM for sequencing into HiSeq-2500 (Illumina, San Diego, CA).