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Sample GSM3719280

Query DataSets for GSM3719280

Status

Public on Apr 11, 2022

Title

PW030-705 (NovaSeq)

Sample type

SRA

Source name

Human glioma slice culture treated with RO4929097 (NovaSeq)

Organism

Homo sapiens

Characteristics

drug treatment: RO4929097 50 nM
tissue: glioma slice culture

Treatment protocol

The slices were first rested for 6 hours with the maintenance medium in a humidified incubator at 37℃ and 5% CO2. Then, the medium was replaced with pre-warmed medium containing 5 uM Etoposide (Tocris Bioscience, 100 mM stock) or 50 nM RO4929097 (Selleck Chem, 10 mM stock), or corresponding volume of vehicle (DMSO). Slices were cultured with the treatment medium in a humidified incubator at 37℃ and 5% CO2 for 18 hrs before being collected for dissociation.

Growth protocol

Acute slices were generated from a surgical resection specimen collected from an adult patient with radiographic and intraoperative histopathological characteristics consistent with primary HGG, (the final diagnosis was Glioblastoma, IDH-wildtype, WHO grades IV). The surgically excised specimen was immediately placed in a sterile 50mL canonical tube ¾ filled ice-cold high-sucrose low-sodium artificial cerebrospinal fluid (ACSF) solution containing 210 mM sucrose, 10 mM glucose, 2.5 mM KCl, 1.25 mM NaH2PO4, 0.5 mM CaCl2, 7 mM MgCl2 and 26 mM NaHCO3, and were kept on ice for transportation (transit time was approximately 10 minutes from operating room to laboratory). Preparation of ex vivo tissue slices was modified from methods described previously (Stoppini et al. 1991 & Gähwiler et al. 1997 Köhlinge et al. 1999, Shimizu et al. 2011, and Ting et al. 2018). Briefly, the tissue specimen was placed in ice-cold ACSF solution and 500 um slices were generated using a tissue chopper (McIlwain TC752). The slices were immediately transferred to the ice-cold ACSF solution in 6-well plates using a sterile plastic Pasteur pipette half filled with ice-cold ACSF solution followed by a 15 minutes recovery in ACSF to reach room temperature. Slices were then placed on top of a porous membrane insert (0.4 um, Millipore). The membrane inserts were placed into 6-well plates containing 1.5 ml maintenance medium consisted of F12/DMEM (Gibco) supplemented with N-2 Supplement (Gibco) and 1% penicillin/streptomycin (Sigma). To ensure proper diffusion into the slice, culture medium was placed under the membrane insert without bubbles. A drop of 10 ul of culture medium was added directly on top of each slice to prevent the slice surface from drying.

Extracted molecule

polyA RNA

Extraction protocol

Collected slices were dissociated using Adult brain dissociation kit (Miltenyi Biotec) on gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) according to the manufacturer’s instructions.
Dissociated cells from each slice were subjected to microwell-based single-cell RNA-seq (Yuan & Sims 2016) as previously described (Yuan et al. 2018) with modifications to the reverse transcription step and the sequencing method. Once the RNA-capture step was finished, sealing oil was flushed out of the devices by pipetting 1ml wash buffer supplemented with 0.04 U/μl RNase inhibitor (Thermo Fisher Scientific) and then beads were extracted from the device and resuspended in 200 ul of reverse transcription mixture. Bead-suspensions were divided into 50ul aliquots and placed into PCR tubes (Corning) followed by incubation at 25°C for 30 min and at 42°C for 90 min in a thermocycler. For sequencing, we pooled all libraries derived from the same donor, each of which was barcoded with a unique Illumina sample index. The pooled library sequenced on 1) an Illumina NextSeq 500 with an 8-base index read, a 21-base read 1 containing cell-identifying barcodes (CB) and unique molecular identifiers (UMIs), and a 63-base read 2 containing the transcript sequence, and 2) an Illumina NovaSeq 6000 with an 8-base index read, a 26-base read 1 containing CB and UMI, and a 91-base read 2 containing the transcript sequence.\

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

PW030-705.matrix.txt.gz

Data processing

Raw data obtained from the Illumina NextSeq 500 was trimmed and aligned as described in Yuan et al (2018). For each read with a unique, strand specific alignment to exonic sequence, we constructed an address comprised of the CB, UMI barcode, and gene identifier. Raw data obtained from the Illumina NovaSeq 6000 was first corrected for index swapping to avoid cross-talk between sample index sequences as described in Szabo et al. 2019. The resulting address file only contained the addresses from reads with corresponding single index. We combined the addresses from the NextSeq 500 and the corrected addresses from the NovaSeq 6000 to generate a digital gene expression matrix and filter CBs as described previously (Yuan et al. 2018). Briefly, reads with the same CB, UMI and aligned gene were collapsed and sequencing errors in the CB and UMI were corrected to generate a preliminary matrix of molecular counts for each cell. Then we removed CBs 1) with excessive (> 10%) molecules expressed from mitochondrial genome, 2) where the ratio of molecules aligning to whole gene bodies (including introns) to molecules aligning exclusively to exons was >1.5, and 3) for which the average number of reads per molecule or average number of molecules per gene deviated by >2.5 standard deviations from the mean for a given sample.
Genome_build: GRCh38
Supplementary_files_format_and_content: Tab-delimited text files that contain the gene expression counts of each individual cell.

Submission date

Apr 11, 2019

Last update date

Apr 11, 2022

Contact name

Peter A Sims

E-mail(s)

pas2182@columbia.edu

Organization name

Columbia University

Street address

3960 Broadway, Lasker 203AC