 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 08, 2017 |
| Title |
CS-Seq_A549_siRNA_00hpi_siDIS3-1 |
| Sample type |
SRA |
| |
|
| Source name |
CS-Seq_A549_siRNA_00hpi_siDIS3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A549
cell type: adenocarcinomic human alveolar basal epithelial cells
viral strain: NA
treatment: siDIS3
timepoint: 00 hours post-infection
genome build for data mapping: hg38(GRCh38) with GENCODE v23 annotations
|
| Treatment protocol |
Transfection was performed using the Lipofectamine RNAiMAX transfection reagents according to the manufacturer’s instructions (Invitrogen). Cells were transfected with siRNA pools (all from Dharmacon) targeting the genes encoding human EXOSC10 or DIS3, or with a control non-targeting pool, at a final concentration of 50 nM. Cells were used 48 hours after transfection. A549 cells were infected with Influenza A/Puerto Rico/8/1934 H1N1Viral at a multiplicity of infection (MOI) of 3 and cells were analyzed at 4 hours post infection.
Transfection was performed using the Lipofectamine RNAiMAX transfection reagents according to the manufacturer’s instructions (Invitrogen). Cells were transfected with siRNA pools (all from Dharmacon) targeting the genes encoding human EXOSC10 or DIS3, or with a control non-targeting pool, at a final concentration of 50 nM. Cells were used 48 hours after transfection. A549 cells were infected with Influenza A/Puerto Rico/8/1934 H1N1Viral at a multiplicity of infection (MOI) of 3 and cells were analyzed at 4 hours post infection.
B cells were treated with 100 nM tamoxifen for 3 days in order to induce the conversion event. B cells were infected with Influenza A/Puerto Rico/8/1934 H1N1Viral at a multiplicity of infection (MOI) of 3 and cells were analyzed at 6 hours post infection.
Transfection of plasmid DNA was achieved using Lipofectamine 3000 (ThermoFisher Scientific). A549 cells were used 36 hours after transfection. A549 cells were infected with Influenza A/Puerto Rico/8/1934 H1N1Viral at a multiplicity of infection (MOI) of 3 and cells were analyzed at 6 hours post infection.
|
| Growth protocol |
A549 cells (adenocarcinomic human alveolar basal epithelial cells) were originally obtained from the American Type Culture Collection (ATCC). Cells were maintained in culture at 37°C with 5% CO2 in Dulbecco’s minimal essential medium (DMEM, Gibco, Life Technologies) supplemented with 2 mM glutamine (Life Technologies), 10% fetal bovine serum (FBS; Hyclone), penicillin (100 U/ml; Life Technologies), and streptomycin (100 μg/ml; Gibco, Life Technologies)
We utilized the conditional system developed by (Pefanis et al., 2014). In short, this mouse model contains a conditional inversion (COIN) allele of Exos¬c3, allowing conditional ablation of RNA exosome function using tissue-specific or inducible Cre recombinase. Cre-mediated ablation of Exosc3 leads to concomitant green fluorescent protein (GFP) reporter induction from the Exosc3 locus. As such, there are two mouse types: COIN/+ (heterozygous for the conditional ablation), and COIN/COIN (homozygous for the conditional ablation). Spleens were obtained from each mouse type, and single cell suspensions were prepared by pressing the tissues through a 70 μm cell strainer, followed by homogenization using a 20g syringe. B cells were purified by negative selection using a MACS column (Miltenyi Biotec) according to the manufacturer's instructions, and then resuspended in DMEM at 500,000 cells/mL in 6-well plates. Cells were growth-stimulated with 20 ng/mL IL-4 (404-ML-010, R&D Systems) and 5 ug/mL purified anti-CD40 (Clone HM40-3, BD Biosciences).
A549 cells (adenocarcinomic human alveolar basal epithelial cells) were originally obtained from the American Type Culture Collection (ATCC). Cells were maintained in culture at 37°C with 5% CO2 in Dulbecco’s minimal essential medium (DMEM, Gibco, Life Technologies) supplemented with 2 mM glutamine (Life Technologies), 10% fetal bovine serum (FBS; Hyclone), penicillin (100 U/ml; Life Technologies), and streptomycin (100 μg/ml; Gibco, Life Technologies)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Capsnatch sequencing : 1 µg of DNase treated RNA was treated with 5U of Cap-Clip Acid Pyrophosphotase (CellScript) for 1.5hrs at 37°C to remove 5’-cap structures. Immediately after, 5 µl of freshly prepared Sodium Periodate was added to a final concentration of 500mM to block free 3’-OH ends of RNA. After incubating for 1.5hrs at 4°C the reaction was stopped by adding 1/10 volume of 1M L-lysine and incubating at room temperature for an additional 10 minutes. Next, RNA was purified with 1.8X volume of AMPure XP beads (Beckman Coulter), and 10 µM barcoded RNA adapters (5'- GUU CAG AGU UCU ACA GUC CGA CGA UCN NNX XXX XXN NN -3’, Bioo Scientific; N=randomized bases; X=barcode adapter) were ligated to the 5’ ends of RNAs in an overnight (~16-18hrs) reaction at 16°C containing 5U of T4 RNA ligase, 10 µM ATP, and 1 µl of RNAse Inhibitor (Promega, N2618). Adapter-ligated RNA was purified with 1.8X volume of AMPure XP beads, and up to six 1 µg samples with distinct 5’ adapter barcodes were pooled together. Ribosomal RNAs were removed from pooled samples using the Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat) (Illumina) according to the manufacturer’s protocol, using 4 µls of Reaction Buffer and 10 µls of rRNA Removal Solution. rRNA depleted samples were purified with 1.6X volume AMPure XP beads and reverse-transcribed using by hybridizing 10 µM of a custom 3’ primer (5'-AGA CGT GTG CTC TTC CGA TCT N*N*N*N*N*N*-3',
Bioo Scientific, N*=randomized bases) for 2 minutes at 65°C. Then, 25mM of dNTP Mix (Thermo Scientific), 1 µl of RNasin® Plus RNase Inhibitor (Promega), 5X First Strand Buffer, 200mM of DTT, and SuperScript® III Reverse Transcriptase (Invitrogen) were added to the reaction and incubated in a thermocycler for 25°C for 10 minutes, 42°C for 50 minutes and 70°C for 15 minutes. Next, cDNAs were purified with 1.8X volume AMPure XP beads, followed by PCR amplification using Kapa HiFi HotStart Ready Mix, 25µM SR forward primer (5'-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG TTC AGA GTT CTA CAG TCC G-s-A-3', Bioo Scientific) and a reverse indexed primer (5’-CAA GCA GAA GAC GGC ATA CGA GAT XXX XXX GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC*T-3’, IDT) for 20 cycles. PCR products were purified using 1.8X AMPure XP beads fragments of 200-400bp were size-selected using BluePippin 2% M1 gels (Sage Scientific). Following library validation on the Agilent Bioanalyzer samples were sequenced on the Illumina HiSeq 2500 platform in a 100 bp single-end read run format.
A549 cells were washed 3X in cold PBS, and total RNA was extracted using Trizol reagent.
B cells were washed 3X in cold PBS, and total RNA was extracted using Trizol reagent.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
library strategy: Capsnatch sequencing
For Capsnatch-Seq: Due to the design of our 5’ RNA adapter, each capsnatch-Seq read starts with 12 adapter-derived nucleotides, consisting of 3 random nt, followed by a 6-nt unique barcode sequence, and another 3 random nts. Demultiplexing and removal of these 5’ adapter sequences was done using custom scripts, allowing up to two barcode base mismatches and no insertions/deletions. Remaining sequences were additionally trimmed at the 3’-end after reaching a base with a phred quality score lower than 10, or after encountering 15 bases with a phred score lower than 28. Finally, 3' Illumina adapter sequences were removed using cutadapt with a minimal overlap of 6bp and allowing for an adapter error rate of 15%. Reads less than 50nt in length after quality and adapter trimming were removed from further analysis. To identify uniformly host- and virus-derived 5’ RNA sequences, full-length adapter-trimmed reads were mapped to the human (hg38) and viral (IAV) reference genomes using TopHat 2.0.12 and Bowtie 1.0.1 with the corresponding gene annotations (GRCh38/Gencode-v23 for the human genome), requiring a minimal anchor size of 10 nucleotides and using default settings for all other arguments. To identify hybrid viral mRNA sequences, the first 20 nucleotides of each read were first masked prior to mapping to viral reference genomes using the same strategy. Hybrid reads were then identified as those masked reads mapping to the positive strand of a viral genome segment, with a start coordinate less than 20bp from the segment start.
For Directional RNA-Seq: Read sequences were trimmed at the 3’-end after reaching a base with a phred quality score lower than 10, or after encountering 15 bases with a phred score lower than 28, using custom scripts. Next, 3' Illumina adapter sequences were removed using cutadapt with a minimal overlap of 6 bp, allowing for an adapter error rate of 15%. Reads less than 50 nt (for paired-end 100 nt reads) in length after quality and adapter trimming were removed from further analysis. Full-length adapter-trimmed reads were mapped to the human (hg38) and viral (IAV) reference genomes using STAR v2.5 with the corresponding gene annotations (Gencode GRCh37/V23 for the human genome), using detection of chimeric alignments with a minimum mapped length and a minimum chimeric overhang junction of 15 nt, and default settings for all other arguments. Total mapped fragment (i.e. paired-end read) counts per gene were determined using featureCounts with default settings
Genome_build: Human: GRCh38/Gencode-v23 annotations
Genome_build: IAV: A/Puerto Rico/8/1934 H1N1
Genome_build: Murine: mm10(GRCm38)/ENSEMBL v75 annotations.
Supplementary_files_format_and_content: Tab-separated text files summarizing for each gencode transcript the number of host-mapped or viral segment reads. One column shows raw counts obtained from featureCounts, second column containes FKPM normalized counts.
|
| |
|
| Submission date |
Mar 15, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Zuleyma Peralta |
| E-mail(s) |
[email protected]
|
| Phone |
212-824-9070
|
| Organization name |
Icahn School of Medicine at Mount Sinai
|
| Department |
Genetics and Genomic Sciences
|
| Lab |
Bakel
|
| Street address |
1470 Madison Ave Rm 8-302
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10029 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE96677 |
The nuclear RNA exosome is co-opted to enhance host:viral RNA hybrids that propel influenza virus ribogenesis and infectivity |
|
| Relations |
| BioSample |
SAMN06605662 |
| SRA |
SRX2643393 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2538143_CS-Seq_A549_siRNA_00hpi_siDIS3-1.counts.txt.gz |
160.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
