Hiseq pe cluster kit v4

The libraries were pooled at equimolar concentrations and diluted prior to loading onto the flow cell of the Illumina cBot cluster station. The libraries were extended and bridge amplified to create sequence clusters using the Illumina HiSeq PE Cluster Kit v4 and sequenced on an Illumina HiSeq Flow Cell v4 with 50-bp paired-end reads plus index read using the Illumina HiSeq SBS Kit v4. Real-time image analysis and base calling were performed on the instrument using the HiSeq Sequencing Control Software version 2.2.58. All samples had a minimum of 43,303,826 passed-filter single-end reads. The sequences aligned at an average of 72% ± 2% (SD) efficiency to the reference genome.

Samples from 39 lymphoid cancer families, including 39 early onset cases and relatives, were part of a batch of 92 samples subject to whole exome sequencing, performed using an Illumina HiSeq2500 instrument in High Output mode with HCS (v2.2.68) at the Genome Sciences Centre (Vancouver, British Columbia, Canada). DNA extraction for 37 early onset cases was from blood; for two cases, saliva samples were used. Briefly, library preparation involved shearing of DNA using Covaris E-Series (Covaris, E210) to an average fragment size of 250 bp and using NEB Paired-End Sample Prep Kit (NEB, E6000B-25B; protocol as per kit version 1.1) for phosphorylation, d-A tailing, adaptor ligation, and PCR enrichment of sheared samples. Capture was performed with Agilent SureSelect v5 + UTR kit capture probes using four libraries per capture, for a total of 23 captures. The protocol was carried out according to the Agilent SureSelect Target Enrichment System for Illumina Multiplexed Sequencing. Amplification was performed using the HiSeq PE Cluster Kit v4 (Illumina, PE-401-4001) and cBot Cluster Station. Sequencing was performed using the HiSeq SBS Kit v4 (Illumina, FC-401-4003) and the Illumina HiSeq2500 System, generating 125 bp reads.

RNA samples of the hvCAS were harvested and extracted by Trizol as mentioned before. DNA libraries were prepared by KAPA Stranded mRNA-Seq Kit (Kapa Biosystems). HiSeq PE Cluster Kit v4 with cbot and HiSeq SBS Kit v4 (Illumina) was used for Pair-End 101bp sequencing by HiSeq 1500 (Illumina). Manufacturers’ protocols were followed. Sequencing reads were first filtered for the adapter sequence and low-quality sequence, and only reads with a read length ≥40 bp were retained. The rRNA-filtered raw reads showed alignment (human reference genome GRCh37 using TopHat) and transcriptome assembly (Cufflink). For each collection of gene sets, the gene sets with FDR-corrected p-values less than 0.20 were deemed significantly varied (enriched or depleted) ones.

RNA quality was checked using the Bioanalyzer (Agilent). For RNA extracted from tumour epithelial cells or tumour mesenchymal cells, indexed cDNA libraries were obtained using the TruSeq Stranded mRNA Sample prep kit (Illumina) according to the manufacturer’s recommendations. The multiplexed libraries (11 pM) were loaded and sequences were produced using a HiSeq PE Cluster Kit v4 and TruSeq SBS Kit v3-HS (250 cycles) on the HiSeq 1500 (Illumina) system. Approximately 8 million paired-end reads per sample were mapped against the mouse reference genome (GRCm38.p4/mm10) using STAR software to generate read alignments for each sample. Annotations Mus_musculus.GRCm38.84.gtf were obtained from https://ftp.ensembl.org/. After transcripts were assembled, gene-level counts were obtained using HTSeq and normalized to 20 million aligned reads. Fold change (FC) values were computed on these values between the conditions. The accession number for the RNA-seq reported in this paper is GEO: GSE205985.