GoKawiilHuman glioma samples
Frozen glioma tissue specimens with the IDH1 or IDH2 mutation were collected with informed consent from the following tissue source sites: Saint Joseph’s Hospital, the Luxembourg Institute of Health-NORLUX (LIH-NORLUX), the MD Anderson Cancer Center (MDACC), Seoul National University (SNU) Hospital and the Pitié-Salpêtrière Hospital. Sample collection was approved by each tissue source site’s Institutional Review Board (IRB). The IRB protocol numbers of respective institutes are as follows; MDACC, 2012-0441; LIH-NORLUX, 201201/06; SNU, H-2004-049-1116; Pitié-Salpêtrière Hospital, 96-900; and Saint Joseph’s Hospital, 2020-NHSR-0084. IDH mutation status and chromosome 1p and 19q co-deletion status were obtained from pathology reports and confirmed by DNA-seq in this study when available. Detailed clinical information, including patient sex, age at diagnosis and treatment information, is provided in Supplementary Table 1. Astrocytoma samples with evidence of CDKN2A homozygous deletion were assigned grade 4 status in accordance with the 2021 WHO diagnostic criteria1. Adjacent fragments from each glioma sample were aliquoted for single-nucleus assays and bulk tissue nucleic acid isolation when sufficient material was available.
Tissue processing and nucleus isolation
Isolation of nuclei from frozen glioma samples was performed by three separate laboratories using two different protocols. Frozen glioma samples from the same patient were always processed together. Laboratory 1 and laboratory 2 processed the cohorts from MDACC and Pitié-Salpêtrière Hospital using a previously described protocol46. In brief, frozen samples were thawed and mechanically dissociated in 0.49% CHAPS with salts and Tris (ST) buffer (10 mM Tris-HCL pH 7.5, 146 mM NaCl, 1 mM CaCl 2 and 21 mM MgCl 2 ). Single-nucleus suspensions were filtered using a 40 μm strainer, centrifuged at 500g for 5 min and resuspended in ST buffer with 0.01% BSA (Sigma). Final nucleus suspensions were stained by Trypan blue and counted using a haemocytometer. The number of nuclei was then determined for use in a 10x Genomics workflow (that is, targeted nucleus recovery) or a FACS-sorting for Smart-seq2 workflow.
Laboratory 3 processed the cohorts from LIH-NORLUX, SNU and Saint Joseph’s Hospital with a protocol using EZ lysis buffer (Millipore Sigma). In brief, frozen samples were thawed and mechanically dissociated in Nuclei EZ lysis buffer via dounce homogenization. The solutions were incubated on ice for 5 min and mixed 1–2 times during incubation. Single-nucleus suspensions were filtered through a 70 μm strainer and centrifuged at 500g for 5 min at 4 °C, resuspended in Nuclei EZ lysis buffer and incubated on ice for 5 min. The solutions were centrifuged at 500g for 5 min at 4 °C and resuspended 3 times in 1% BSA and 0.2 U μl–1 RNase inhibitor and PBS buffer. The final suspension of nuclei was stained with DAPI, filtered through a 40 μm strainer and counted using a Countess II automated cell counter (Thermo Fisher Scientific). The number of nuclei was then determined for use in 10x Genomics 3′ RNA or Multiome workflow.
Bulk DNA-seq
Bulk DNA-seq was performed in a tissue source site-specific manner. For samples from the MD Anderson Cancer Center, DNA was extracted from each frozen glioma sample and blood sample corresponding to the patients using a DNeasy Blood & Tissue kit (Qiagen). The genomic DNA (100–250 ng) was acoustically sheared using an ultrasonicator (Covaris), targeting 150 bp fragments. Library preparation was performed using a KAPA HyperPrep kit (KAPA Biosystems) followed by clean-up using AMPure XP beads (Beckman Coulter). Exome capture was performed using a custom exome bait (manufactured by Twist Biosciences to The Broad Institute’s specification). Captured libraries were sequenced with 150-bp paired-end sequencing on a NovaSeq 6000 (Illumina). For the samples from Pitié-Salpêtrière Hospital, after the DNA was fragmented using a LE220 ultrasonicator (Covaris) and size selected, library preparation and capture were performed using a Twist Human Core Exome kit (Twist Bioscience) according to the manufacturer’s protocol. Sequencing was performed on a NovaSeq 6000 (Illumina). WGS data were generated for frozen samples from Saint Joseph’s Hospital, SNU and LIH-NORLUX. In brief, DNA was extracted from each glioma sample using an AllPrep DNA/RNA Minikit (Qiagen) for samples with sufficient tumour tissue and matched normal blood when it was available. DNA was sheared to 400 bp using a LE220 ultrasonicator (Covaris) and size-selected using AMPure XP beads (Beckman Coulter). Whole-genome libraries were prepared and sequenced with 150-bp paired-end sequencing on a NovaSeq 6000 (Illumina). Whole-exome sequencing was additionally performed for Saint Joseph’s Hospital samples using Agilent SureSelect Human All Exon v7 capture kit, followed by 150-bp paired-end sequencing on a NovaSeq 6000 (Illumina). The WGS data for the SNU cohort was previously reported14.
snRNA-seq and ATAC–seq
A 10x Chromium Single Cell 3′ Reagent kit v.3 (10x Genomics) was used according to the manufacturer’s protocol. In brief, nuclei were loaded on a Chromium chip (10x Genomics) with a target cell recovery of 6,000–8,000 nuclei and processed in a Chromium Controller. Single-nucleus samples were partitioned into gel beads-in-emulsion (GEMs), followed by RNA reverse transcription with barcoding. Libraries were created by breaking GEMs and pooling barcoded fractions, cDNA amplification, fragmentation and attachment of a sample index and adapter and sequenced on a Nextseq500 or Novaseq (Illumina). For three samples in the Saint Joseph’s Hospital cohort, a 10x Chromium Single Cell 3′ Reagent kit v.3 was used. For LIH-NORLUX, SNU and the remainder of the Saint Joseph’s Hospital samples, nuclei were loaded on a Chromium chip with a target cell recovery of 6,000 nuclei for 10x single-cell Multiome ATAC and gene expression according to the manufacturer’s protocol.
Nucleus sorting for Smart-seq2
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