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| Status |
Public on Jun 30, 2017 |
| Title |
H929 cells expressing FAM46CD90A,D92AGFP for 72 hrs rep4_total RNA |
| Sample type |
SRA |
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| Source name |
H929 cells expressing FAM46CD90A,D92AGFP for 72 hrs
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| Organism |
Homo sapiens |
| Characteristics |
cell line: H929
cell type: Multiple myeloma cell line
tranduced with: lentiviruses carrying FAM46CD90A,D92AGFP for 72 hours
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| Treatment protocol |
MM cells were transduced with lentiviruses carrying FAM46CWTGFP or FAM46CD90A,D92AGFP For lentivirus production, 2.6 x 106 HEK-293T cells were seeded into 10 cm dishes 24 hours prior to transfection in 10 mL of DMEM supplemented with 10% FBS (Invitrogen) and penicillin/streptomycin solution (Sigma). The lentiviruses encoding the gene of interest (GOI) were introduced to the packaging cells by calcium phosphate co-transfection with 8.6 µg of a GOI-containing vector HIV-SFFV and components of 2nd generation of packaging vectors, namely, 8.6 µg of psPAX2 packaging vector and 5.5 µg of pMD2.G envelope vector. The plasmids were resuspended in 450 µl of 300 mM CaCl2 and added dropwise to 450 μl of 2 × concentrated HEPES-buffered saline (280 mM NaCl, 20 mM HEPES, 1.5 mM Na2HPO4, 10 mM KCl and 12 mM D-glucose pH 7.2) by vortexing. The precipitate was immediately added to the cell culture medium with gentle swirling. The medium was replaced with 6 mL of fresh DMEM 16 hours post transduction. Virus-containing supernatants were collected 24 hours later, centrifuged for 3 min at 300 x g, and filtered through 0.45 μm low-protein binding filters (Millipore) Then, the supernatants were concentrated 10 × by centrifugation (3000 x g, 16 hours, 4°C). The titer of viral particles was estimated using a Lenti-X p24 Rapid Titer Kit (Clontech) according to the manufacturer’s protocol. Multiplication of infection (MOI) was calculated by dividing the vector titer for the number of cells transduced. Efficiency of transduction was determined by flow cytometry for GFP-expressing cells using an Accuri C6 benchtop cytometer (BD Biosciences). Additional staining with 2.5 µg/mL propidium iodide (Sigma-Aldrich) in PBS was performed to distinguish dead cells. If needed, to select GOI-expressing cells, sorting for GFP-positive cells using the Aria III cell sorter (BD Biosciences) was performed.
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| Growth protocol |
Multiple myeloma cell line SKMM1 and H929 (ATCC; CRL-9068) were cultured in RPMI 1640 ATCC’s modified (Invitrogen) supplemented with 10% FBS (Invitrogen) and penicillin/streptomycin (Sigma; P4083) at 37°C in 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was extracted and RNA-seq libraries were prepared. Replicate #4.
RNA isolation was performed with Trizol reagent (Invitrogen, 15596) according to the manufacturer’s instructions. Poly(A) fractionation was performed as previously described with the following modifications [Meijer, H.A. et al. A novel method for poly(A) fractionation reveals a large population of mRNAs with a short poly(A) tail in mammalian cells. Nucleic Acids Res 35, e132 (2007)]. In brief, 80 µg of total RNA was mixed with 400 µl GTC buffer (4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7.1, 2% β-mercaptoethanol) and 750 pmoles of biotinylated 5’biotin –TEG-oligo(dT)25 (Future Synthesis). 850 µl of “dilution buffer” (6× SSC, 10 mM Tris–HCl, pH 7.4, 1 mM EDTA, 0.25% SDS, 1% β-mercaptoethanol) was added, the mixture was incubated at 70°C for 5 minutes, and then it was centrifuged at 12 000 rpm for 10 min at RT. The resulting supernatant was mixed with 100 µl of M280 beads (Invitrogen; 60210) washed 3 times with 0.5x SSC buffer. After binding, samples were washed 3 times with 0.5x SSC buffer for 10 minutes. RNA fractions were eluted with decreasing concentrations of SSC buffer (from 0.2x SSC to 0.025x SSC) and the final elution was performed with water. RNA was precipitated and subjected to further analysis.
1 µg of total RNA was treated with Turbo-DNase (Life Technologies) according to the manufacturer’s instructions. Then, rRNA was removed from samples using a Ribo-Zero Kit (Epicentre), according to the manufacturer’s protocol, and samples were spiked-in with external RNA (ERCC RNA Spike-In Mix; Life Technologies). For libraries prepared from poly(A)+ fractions, the ribo-depletion step was omitted. Finally, RNA libraries were prepared using a TruSeq RNA Sample Preparation Kit (Illumina) or a KAPA Stranded RNA-Seq Library Preparation Kit (Kapa Biosystems) according to the manufacturer’s instructions. Quality of libraries was determined by chip electrophoresis performed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
H929_FAM46C_mut_8
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| Data processing |
base caling CASAVA v1.8.2
aligmnent STAR v2.4.0
further processing, filtering, counting, QC - custom scripts emploing samtools v1.2-10, HTSeq v0.6.1, RSeQ v2.6.1, FastQC v0.11.3
differential expression analysis DESeq2 v1.8.2
Genome_build: hg38
Supplementary_files_format_and_content: bigWig files (corected for total library size) and count tables (FPKM_count.py or htseq-count output files)
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| Submission date |
Jun 27, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Tomasz M. Kuliński |
| E-mail(s) |
kulatom@gmail.com
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| Phone |
225970602
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| Organization name |
International Institute of Molecular and Cell Biology in Warsaw
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| Lab |
Andrzej Dziembowski
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| Street address |
ul. Ks. Trojdena 4
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| City |
Warszawa |
| State/province |
Mazowieckie |
| ZIP/Postal code |
02-109 |
| Country |
Poland |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE83772 |
The FAM46C gene encodes a non-canonical poly(A) polymerase and acts as an onco-suppressor in multiple myeloma |
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| Relations |
| BioSample |
SAMN05296484 |
| SRA |
SRX1880030 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2218716_H929_mut8.F.bw |
596.4 Mb |
(ftp)(http) |
BW |
| GSM2218716_H929_mut8.FPKM.txt.gz |
4.5 Mb |
(ftp)(http) |
TXT |
| GSM2218716_H929_mut8.R.bw |
561.4 Mb |
(ftp)(http) |
BW |
| GSM2218716_H929_mut8.countsGene.txt.gz |
228.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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