Bioanalyzer 2100

GCps were purified from P7 mice as described above. Intact nuclei were isolated (EZ Prep Nuclear Isolation Kit, Sigma-Aldrich) from 50,000 freshly isolated GCps of each genotype, and ATAC-Seq libraries were produced as previously described (44 (link)). Library quality was assessed on a 2100 Bioanalyzer and quantified using the KAPA library quantification kit (Kapa Biosystems). Six libraries were multiplexed (3 per genotype) and 75 bp paired-end reads were sequenced on a HiSeq4000 (Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom). Reads were uniquely aligned to the mouse reference genome (mm10) using Bowtie (version 1.1.1) (66 (link)) with a maximum insert size for valid paired-end alignments of 2,000 and a maximum number of attempts of 200 to match an alignment for 1 mate with an alignment for the opposite mate. Peaks were called in 3 control replicates by comparing them to 3 cko samples and vice versa using MultiGPS (version 0.5) (67 (link)) with the minimum q value for reported peaks set at 0.05 and the mitochondrial genome excluded. The bedtools window function from BEDTools (version 2.17.0) (68 (link)) was used to identify ATAC-Seq peaks within 1,000 bp of differentially expressed genes (DEGs).

Library preparation was performed at the University of Florida’s Interdisciplinary Center for Biotechnology Research (ICBR) Gene Expression Core. RNA integrity was verified using the Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, United States). Of the total RNA, 10 ng were used to construct cDNA libraries with the ClonTech SMART-Seq v4 ultra-low input RNA kit for sequencing (Clontech Laboratories, Inc., Cat#: 634890), according to the manufacturer’s instructions. Briefly, 1st strand cDNA was primed by the SMART-Seq v4 oligonucleotide, which then base-pairs with these additional nucleotides, creating an extended template. The reverse transcriptase then switches templates and continues transcribing to the end of the oligonucleotide, resulting full-length cDNA that contains an anchor sequence that serves as a universal priming site for second strand synthesis. Then, cDNA was amplified with primer II A for 8 PCR cycles. Illumina sequencing libraries were then generated with 150 pg of cDNA using the Illumina Nextera DNA Sample Preparation Kit (Cat#: FC-131-1024) according to the manufacturer’s instructions. Briefly, 150 pg of cDNA were fragmented by tagmentation reaction and adapter sequences were added onto template cDNA by PCR amplification. Libraries were quantitated using both the 2100 BioAnalyzer and qPCR (Kapa Biosystems, catalog number: KK4824).

TRIzol reagent was utilized for extracting total cellular RNA, which was later subject to spectrophotometry and agarose gel electrophoresis (AGE) with the NanoDrop ND-1000 instrument to analyze RNA integrity. Using a KAPA Stranded RNA-seq Library Prep Kit, this study built an RNA library, while a 2100 Bioanalyzer was employed for library quality assessment. Quantification of the library was performed by qRT-PCR. NCBI Gene Expression Omnibus was utilized to import raw RNA-seq information.

High quality RNA (1 μg) was subjected to paired-end sequencing using the HiSeq2500 Illumina platform. Complementary DNA libraries were constructed using mRNA-Seq Sample Prep Kit based on the Illumina guide. Library size distribution was validated using Agilent Technologies 2100 Bioanalyzer and quantified by quantitative PCR (KAPA Library Quant Kits, KAPA biosystem). Four normalized sample libraries were pooled together and loaded to a single lane of an Illumina flow cell. Data analysis was performed at the Vital-IT Systems Biology Division, SIB, Lausanne, and at the Lausanne Branch of the Ludwig Institute for Cancer Research. Differential gene expression between groups was performed with DESeq2^{37 (link)} in R. The genes were ranked based on the fold change between patients with or without PBL neo-epitope recognition and this ranked gene list was inputted into GSEA^{38 (link)} to perform gene set enrichment analysis. For this analysis, the enrichment for all the “Canonical pathways” gene sets (version 5.1) from the “Molecular Signature Database”^{39 (link)} was tested (some results are highlighted in Fig. 1i,j and Supplementary Fig. 2; all results with an FDR below 0.05 are reported in Supplementary Table 3).

Sorted AML blasts were prepared with 10X Genomics Chromium Single Cell ATAC Kit v1.1 according to the manufacturer’s specifications. Samples were uniquely barcoded and quantified using an Agilent BioAnalyzer 2100 or a KAPA qPCR quantification kit. Sample libraries were loaded onto an Illumina NovaSeq and sequenced with 50×8×16×50 read configuration with an average of 25,000 paired end reads per single cell.

Plasma cfDNA from fresh whole blood was extracted using QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocols. The cfDNA concentration and quality were assessed using Qubit 3.0 (Thermo Fisher Scientific, Waltham, MA, USA) and Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA), respectively. The cfDNA libraries for WGS sequencing were prepared using about 20 ng cfDNA as previously described [16 (link)]. Briefly, cfDNA was processed by end repairing, dA tailing and ligation to loop adapter, followed by size selection using AMPure XP beads (Beckman Coulter, High Wycombe, UK). The adaptor‐ligated DNA fragments were amplified by a 14‐cycle PCR using two Illumina p5 and p7 primers. Subsequently, libraries were quantified with the KAPA Library Quantification kit (KAPA Biosystems, Wilmington, MA, USA) and library fragment size was determined by Bioanalyzer 2100. WGS sequencing was then conducted on the Illumina HiSeq X10 platform using a paired‐end 150‐bp protocol.