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Sample GSM5934734

Query DataSets for GSM5934734

Status

Public on Dec 31, 2023

Title

LC_5

Sample type

SRA

Source name

TOV-21G

Organism

Homo sapiens

Characteristics

cell type: TOV-21G, derived from human ovarian cancer
treatment: NA
strain: NA
collection: LC-seq

Treatment protocol

For some experiments, TOV-21G cells were cultured in the Complete Growth Medium added with a IκB kinase IKK inhibitor, IKK-16 IKK Inhibitor VII Sigma-Aldrich at 1 µM for 72 hours before RNA collection.

Growth protocol

Coverslips Fisher Scientific, 12-545-80 were coated with Poly-L-Lysine PLL by incubation in PLL solution 0.01% for 30 minutes at 37°C. TOV-21G ATCC, CRL-11730 cells were grown for 48 hours on cover slips in the Complete Growth Medium [a 1:1 mixture of MCDB 105 1.5 g/L sodium bicarbonate and MCDB 199 2.2 g/L sodium bicarbonate added with fetal bovine serum at a final concentration of 15%] for RNA collection.
Mice were deeply anesthetized with isoflurane Halocarbon. A block of brain containing motor cortex MCx was removed and coronal slices 300 μm thick were cut on a vibrating microtome Leica in ice-cold physiological solution ACSF. The solution contained the following in mM: 130 NaCl, 3 KCl, 1.25 KH2PO4, 20 NaHCO3, 10 glucose, 1.3 MgSO4, 2.5 CaCl2 pH 7.35-7.4, in 95% O2-5% CO2. All solutions were made with nuclease-free, de-ionized, not DEPC-treated water Ambion AM 9938. Slices were incubated for 1 hour at 30°C, then kept at room temperature. For electrophysiological extraction of RNA, slices were held in a chamber on a motorized stage microscope Olympus BX61WI, Gibraltar Burleigh and superfused with ACSF. The location of motor cortex MCx was identified by 1.5-2.0 mm rostral to the start of the hippocampus.

Extracted molecule

total RNA

Extraction protocol

For RNA extraction, cultures and slices were held separately in a chamber on a motorized stage on a motorized microscope Olympus BX61WI, Gibraltar Burleigh Thorlabs. Cultures were superfused 2-4 ml/min in a bath solution of in mM: 145 N-methylglucamine NMG, 2 CaCl2, 1 MgCl2, 10 HEPES, 60 glucose pH 7.3, osmolality 300, in 95% O2-5% CO2 . All procedures were performed with a Multiclamp 700B patch-clamp amplifier Molecular Devices, LLC and using borosilicate glass pipettes with filament, OD 1.5 mm, ID 1.10 mm, Sutter and the input resistance was in the range of 3-9 MΩ. RNA was extracted with a 1440A Digidata AD board Molecular Devices, LLC under pClamp v10. Electrodes pipettes were filled with in mM: 100 KCl, 35 N-methylglucamine NMG, 10 EGTA, 1 CaCl2, 4 MgCl2, and 10 HEPES pH 7.3, 290 mOsm for the RNA extraction from the TOV-21G cells. Electrodes were filled with in mM: 130 K-gluconate, 10 KCl, 10 HEPES, 10 EGTA, 2 MgCl2, 2 Na-ATP, 0.2 Na-GTP, and 5 glutathione pH 7.3, 280-290 mOsm for the RNA extraction from the Thy1-YFP L5 neurons. For the RNA extraction from the Thy1-GCaMP6f L2/3 neurons, electrodes were filled with in mM: 110 K-gluconate, 4 KCl, 4 NaCl, 0.2 CaCl2, 10 HEPES, 1.1 EGTA, 2 Mg-ATP, 1 MgCl2, and 5 glutathione pH 7.2-7.3, 300 mOsm. Membrane voltages were corrected for an estimated liquid junction potential of about 10 mV. Perforation was performed using -escin antibiotics 50 M from a stock solution 25 mM respective for each cellular population. All solutions were made with nuclease-free, de-ionized, not DEPC-treated water Ambion AM 9938. Lucifer yellow 50 mM was added to the electrode intracellular working solution for all cellular populations. The intercellular working solution including the antibiotics and dye was prepared fresh daily and used at room temperature and were backfilled into the electrodes for the patch-clamp experiments. Holding potentials for TOV-21G cells and cortical neurons were -80 mV and -70 mV, respectively. Electrodes were made by a PP-830 Narishige puller and positive pressure was applied as the pipettes were advanced to the cell of interest. The leak current Ilk was then used for the holding current in the current-clamp protocol and it was noted before and after the current was injected in all cellular populations. Seven current injection steps of 5 pA each was applied for a duration of 500 ms per step for the RNA extraction. Following RNA extraction, electrodes were slowly retracted while leaving the cell intact in its native environment, and the pipette solution was collected for sequencing. Each sample tube was prefilled with 2 µL of IGEPAL working mixture per tube, and positive pressure was applied to eject the internal contents of the electrode into a sample tube. The IGEPAL working mixture contained 65 µL nuclease-free de-ionized water Ambion, AM9938, 25 µL RNaseOUT™ Recombinant Ribonuclease Inhibitor ThermoFisher, 10 µL IGEPAL CA-630 Alfa Aesar, J61055 and was stored in 4°C for up two weeks. RNA was collected in 0.5 mL tubes Protein LoBind Tube 0.5 mL, Eppendorf, 022431064. After RNA extraction, samples were transferred on dry ice and stored at -80°C. After each RNA extraction, the electrode holder and silver-chloride recording electrode were cleaned with 70% ethanol. Cytosolic extraction for all cellular populations was performed by first doing whole-cell patch-clamp, then applying negative pressure light suction to harvest cellular content into IGEPAL working mixture.
The cDNA libraries were prepared using Ovation SoLo RNA-Seq System Tecan Genomics. UMIs are used to eliminate possible PCR duplicates in sequencing datasets and facilitate unbiased gene expression profiling. For some experiments, ERCC RNA Spike-In Mix Thermo Fisher Scientific was included in the library preparation. The processed libraries were assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit Agilent Technologies. The libraries were pooled and loaded onto a TruSeq SBS v3 flow cells on an Illumina HiSeq 2500 Illumina or an S1 flow cell on an Illumina NovaSeq 6000 Illumina , and run for 2X50 cycles according to the manufacturer's instructions. De-multiplexed and adapter-trimmed sequencing reads were generated using Illumina bcl2fastq version 2.18.0.12 allowing no mismatches in the index read.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

G281_8

Data processing

De-multiplexed and adapter-trimmed sequencing reads were generated using Illumina bcl2fastq (released version 2.18.0.12) allowing no mismatches in the index read. R2 reads were generated as UMI sequences that were later used to extract PCR duplicates. R1 and R3 reads were used as paired sequencing reads. To assess the read quality, FastQC was run, and low quality reads were removed using sickle with default setting. BBDuk version 36_49 was used to trim/filter low quality sequences using “qtrim=lr trimq=10 maq=10” option. Next, alignment of the filtered reads to the human reference genome GRCh38 was done using HISAT2 version 2.1.0 applying --no-mixed and --no-discordant options. UMI specific workflow that was developed and distributed by Tecan Genomics was used to extract reads that are free from PCR artifacts i.e., deduplication. The resulting deduplicated reads were summarized to each gene using HTseq-count version 0.11.2 by supplementing Ensembl gene annotation GRCh38.78 and ERCC spike-in genes.
Genome_build: GRCh38
Genome_build: mm9
Supplementary_files_format_and_content: Raw read counts are provided in TS_LC_WC_NC.txt and MouseAB_Rawcount.txt files, respectively.
paired-end (R1 and R3, R2 is UMI)

Submission date

Mar 04, 2022

Last update date

Dec 31, 2023

Contact name

Yuka Imamura Kawasawa

E-mail(s)

yui102@psu.edu

Organization name

Penn State University

Department

College of Medicine, Pharmacology