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Sample GSM8129834

Query DataSets for GSM8129834

Status

Public on May 01, 2024

Title

MGH229-P4-F10-CD45neg, Smart-seq2

Sample type

SRA

Source name

Oligodendroglioma single cell, MGH229-CD45neg

Organism

Homo sapiens

Characteristics

tissue: Oligodendroglioma single cell, MGH229-CD45neg
treatment (idhi): Yes

Extracted molecule

polyA RNA

Extraction protocol

Fresh specimens were mechanically dissociated with a disposable, sterile scalpel and further enzymatically dissociated into single cell suspensions using the enzymatic brain dissociation kit (papain-based) from Miltenyi Biotec as previously reported. Tumor cells were blocked in 1% bovine serum albumin in phosphate-buffered saline solution (1% BSA / PBS). Cell suspensions were subsequently stained for flow cytometry for 30 min at 4 °C using antibodies specific for CD45 [REA747]-VioBlue and CD3 [BW264/56]-PE from Miltenyi Biotec. Cells were washed with cold PBS, and then incubated for 15 min in 1.5 mL of 1% BSA / PBS containing 1 uM calcein AM (Life Technologies) and 0.33 uM TO-PRO-3 iodide (Life Technologies). Sorting was performed with the FACS Aria Fusion Special Order System (Becton Dickinson) using 488 nm (calcein AM, 530/30 filter; CD3-PE, 585/42 filter), 640nm (TO-PRO-3, 670/14 filter), and 405 nm (CD45-VioBlue, 450/50 filter) lasers. Standard, strict forward scatter height versus area criteria were used to discriminate doublets and gate only singleton cells. Viable cells were identified by staining positive with calcein AM but negative for TO-PRO-3. We sorted individual, viable, CD45+CD3- and CD45+CD3+ immune, and CD45- non-immune single cells into 96-well plates containing cold TCL buffer (QIAGEN) with 1% beta-mercaptoethanol. Plates were frozen on dry ice immediately after sorting and stored at -80 °C prior to whole transcriptome amplification, library preparation and sequencing. Frozen tumor tissue (approximately 5mm x 5mm x 5mm) was placed into a well of a 6-well plate (Corning, Cat. No. CLS3516-50EA) containing 1 ml of CST buffer and processed on ice. Tumor tissue was chopped for 10 minutes using Noyes Spring Scissors (Fine Science Tools, Cat. No. 15514-12). The sample was then filtered through a 40 um cell strainer (Fisher Scientific, Cat. No. 22363547) and the well and filter were washed with an additional 1 ml of CST buffer. The total volume was then brought up to 5 ml with 3 ml of ST buffer and the sample was centrifuged in a 15 ml conical tube at 4°C for 5 minutes at 500 g. The supernatant was removed and the pellet was resuspended in 100 ul ST buffer. The sample was then filtered through a 35 um cell strainer (Falcon, Cat. No. 352235). Nuclei suspension was counted using disposable C-Chip Hemocytometers (INCYTO, Cat. No. DHCN012). Single-nuclei were loaded on 10x genomics or stained by Ruby (ThermoFisher, V10309) and sorted into 96 well plates. IdhR132H mouse glioma cells were cultured in the presence of 1 μM AG-881or vehicle (DMSO control) for > 2 weeks. The treated cells were washed with cold PBS and then stained with 1 uM calcein AM (Life Technologies) and 0.33 uM TO-PRO-3 iodide (Life Technologies) for 30 min at 4°C. Sorting was performed with the FACS Aria Fusion Special Order System (Becton Dickinson) using 488 nm (calcein AM, 530/30 filter) and 640nm (TO-PRO-3, 670/14 filter). Side scatter (SSC) width versus forward scatter (FSC) area, and Trigger Pulse Width versus FSC criteria were used to discriminate doublets. Calcein AM positive and TO-PRO-3 negative live cells were sorted into 96 wells containing TCL buffer (QIAGEN) for the single-cell multi-omics experiment.
RNA from single cells or single nucleus was first purified with Agencourt RNAClean XP beads (Beckman Coulter). B65Libraries from isolated single cells were generated based on the Smart-seq2 protocol (Picelli 2014) with the following modifications.oligo-dT were used to prime reverse transcription with Maxima reverse transcriptase and locked TSO oligonucleotide, which was followed by 20 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) with subsequent Agencourt AMPure XP bead purification as described. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina) with custom barcode adapters (sequences available upon request). Libraries from 768 cells with unique barcodes were combined and sequenced using a NextSeq sequencer (Illumina). Multi-omics library (DNA methylome and transcriptome) was generated based on the Smart-RRBS protocol (Chaligne et al, 2021).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 2000

Description

Smartseq2_Oligodendroglioma_processed_TPM.tsv

Data processing

We processed Smart-seq2 data from raw reads to gene expression matrices as previously described in Neftel et al, 2019. We used bcl2fastq to generate demultiplexed FASTQ files, and aligned the resulting paired-end scRNA-seq reads to the reference transcriptome using Bowtie (v0.12.7). Statistical analysis was done using R version 4.0.1. We merged the gene expression levels which were calculated by RSEM48 for MGH170 and MGH229 with the ones calculated for the reference samples from Tirosh 2016 (downloaded from https://singlecell.broadinstitute.org/single_cell). Analysis was done mainly using the R package scandal which is freely available at https://github.com/dravishays/scandal. Gene expression levels were quantified as TPM (transcript-per-million reads).
Assembly: GRCh38 for Homo sapiens, mm10 for Mus musculus
Supplementary files format and content: tsv

Submission date

Mar 06, 2024

Last update date

May 01, 2024

Contact name

Masashi Nomura

E-mail(s)

masashin0914@hotmail.com

Organization name

Massachusetts General Hospital

Department

Pathology