Nextera xt sample preparation guide

We purified all the amplicons using the Agencourt AMPure XP PCR Purification systems (Beckman Coulter, Pasadena, CA) and quantified the starting DNA library using the Qubit dsDNA BR Assay system (Invitrogen, Carlsbad, CA). The sequencing library construction was performed according to the Nextera XT sample preparation guide (Illumina, San Diego, CA) that uses transposome to fragment and simultaneously adds adapter and barcoding sequences.
The pooled and barcoded libraries were subsequently sequenced using the MiSeq sequencer with v2 kits, which generated 250-base paired-end sequence reads.

The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech) was used to generate first-strand cDNA from 300 pg of totalRNA. Double-stranded cDNA was amplified by long-distance PCR (13 cycles) and purified via magnetic bead clean-up. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide. Next, 150 pg of input cDNA was tagmented (tagged and fragmented) by the Nextera XT transposome. The products were purified and amplified via a limited-cycle PCR program to generate multiplexed sequencing libraries. For the PCR step, 1:5 dilutions of index 1 (i7) and index 2 (i5) primers were used. The libraries were quantified using the KAPA Library Quantification Kit—Illumina/ABI Prism User Guide (Roche Sequencing Solutions). Equimolar amounts of each library were sequenced on a NextSeq 500 instrument controlled by the NextSeq Control Software, version 2.2.0, using two High Output Kits (75 cycles) with the dual index, single-read run parameters. Image analysis and base calling were done by the Real Time Analysis Software, version 2.4.11. The resulting BCL files were converted into FASTQ files with the bcl2fastq software, version 2.18.
Library preparation and RNA-seq were performed at the Genomics Core Facility ‘KFB - Center of Excellence for Fluorescent Bioanalytics’ (University of Regensburg; http://www.kfb-regensburg.de).

First-strand cDNA was generated using SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, Inc., Mountain View, CA, USA). Double-standed cDNA was amplified with LD PCR and purified with AMPure XP beads. Library preparation was constructed conforming to the Illumina Nextera XT Sample Preparation Guide (Illumina, San Diego, CA, USA). In brief, 150 pg of input cDNA was tagmented via Nextera XT transposome. The products were purified and amplified with a limited-cycle PCR program to construct sequencing libraries. The libraries were quantified with the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems, Wobum, MA, USA). Equimolar amounts of each library were pooled for cluster generation on the cBot using the Illumina TruSeq SR Cluster Kit v3. The sequencing run was performed on a HiSeq1000 instrument with TruSeq SBS Kit v3 according to the Illumina HiSeq 1000 System User Guide. Illumina image analysis and base calling were recorded in library base call format (.bcl) and further converted to Fastq files via the CASAVA1.8.2 software.

First-strand cDNA was generated using SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, inc., Mountain View, CA, USA). Double-stranded cDNA was amplified with LD PCR and purified with AMPure XP beads. Library preparation was constructed by conforming to the Illumina Nextera XT Sample Preparation Guide (Illumina, San Diego, CA, USA). In brief, 150 pg of input cDNA were tagmented via Nextera XT transposome. The products were purified and amplified with a limited-cycle PCR program to construct sequencing libraries. The libraries were quantified with the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems, Wobum, MA, USA). Equimolar amounts of each library were pooled for cluster generation on the cBot using the Illumina TruSeq SR Cluster Kit v3. The sequencing run was performed on a HiSeq1000 instrument with TruSeq SBS Kit v3 according to the Illumina HiSeq 1000 System User Guide. Illumina image analysis and base calling were recorded in library base call format (.bcl) before being further converted into Fastq files via the CASAVA1.8.2 software.

RNA extraction, library preparation, and sequencing were performed at the Health Sciences Sequencing Core at UPMC Children’s Hospital of Pittsburgh, as previously described (43 (link)). CD8⁺CD122⁺CD49d^lo TRLs (500–1000 cells/sample) were isolated by FACS into a plate with lysis buffer. cDNA was generated from cell lysates using the SMART-Seq Ultralow Input RNA Kit (Takara Bio), according to the manufacturer’s instructions. The cDNA product was checked by an Agilent Fragment Analyzer system for quality control. The sequencing library was constructed by following the Illumina Nextera XT Sample Preparation Guide. One nanogram of input cDNA was tagged and fragmented (“tagmented”) and amplified using the Illumina Nextrera XT kit (15031942). Sequence libraries of each sample were finally equimolarly pooled and sequenced on an Illumina Nextseq 500 system, using a paired-end 75-bp strategy. All RNA-seq data are deposited in the NCBI GEO database.