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| Status |
Public on Aug 20, 2020 |
| Title |
A549 siMYBL2 rep4 RNA-seq |
| Sample type |
SRA |
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| Source name |
A549 lung adenocarcinoma cell line
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| Organism |
Homo sapiens |
| Characteristics |
tissue: A549 lung adenocarcinoma cell line
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| Treatment protocol |
siRNA knockdown was performed in quadruplicate on A549 cells using 100 nM of ON-TARGETplus siRNA oglionucleotides for human MYBL2 (# L-010444-00-005, Dharmacon - Horizon Discovery, UK), FOXM1 (# L-009762-00-005, Dharmacon - Horizon Discovery, UK) , or non-targeting control (# D-001810-10-05, Dharmacon - Horizon Discovery, UK) mixed with 5X siRNA buffer (# B-002000-UB-100, Horizon Discovery, UK) and transfected using DharmaFECT 1 Transfection reagent (# T-2001-01, Horizon Discovery, UK). Cells were transfected, cultured for 24 hours, and transfected again with the same concentration of siRNA, then were incubated for an additional 24 hours
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| Growth protocol |
Human lung adenocarcinoma A549 cells (ATCC # CRL-185) were grown at 37oC with 5% CO2 in RPMI 1640 (#10-040-CV, Corning, NY, USA) supplemented with 10% FBS (#FBS-500, X&Y Cell Culture, MI, USA) and 100 units/ml of penicillin/streptomycin (formulated by Norris Comprehensive Cancer Center Media Core, CA, USA). Human alveolar epithelial cells (AECs) were isolated from remnant transplant lung from de-identified non-smoking donors as previously described by Marconett CN et al. 2013. AECs were isolated from the samples and plated, after which they differentiated from AT2-like cells to AT1-like cells. Cells used here included freshly isolated AEC (AT2 - D0) cells plated for 4 days (D4) representing cells in an AT2-to-AT1-transitional state, and cells plated for six days (D6), representing AT1-like cells as described by Marconett CN et al. 2013 and Yang C et al. 2018.
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| Extracted molecule |
total RNA |
| Extraction protocol |
ChIP sequencing was performed on D0, D4, and D6 alveolar epithelial cells originally isolated from Donor 1 using antibodies for H3K27ac (catalog #39133, Active Motif, CA, USA) as described previously by Yang C et al. 2018.
ATAC sequencing was performed on D0 alveolar epithelial cells originally isolated from Donor 2 using the protocol from Buenrostro et al. 2015. Briefly, intact nuclei were isolated and incubated with Tn5 transposase (#FC-121-1030, Illumina, CA, USA). The transposed DNA was extracted and was amplified with PCR using NEBNExt High-Fidelity PCR Master Mix (#M0541S, New England Biolabs, MA, USA) and the resulting library was purified using a bead clean with AMPure XP Magnetic Beads (#A63880, Beckman Coulter, CA, USA) and quality control was performed using a BioAnalyzer High-Sensitivity DNA Analysis kit (#5067-4626, Agilent, CA, USA).
For RNA sequencing, RNA was extracted using the Aurum Total RNA Mini Kit (# 7326820, Bio-Rad, CA, USA). cDNA was synthesized using an iScript cDNA Synthesis Kit (# 1708891, Bio-Rad, CA, USA).
ChIP-seq libraries were made was performed at the USC Epigenome center and sequenced using 50bp single-end reads on an Illumina HiSeq 2000 according to the manufacturer’s instructions after performing QC as previously described by Yang C et al. 2018.
ATAC-seq library construction was performed at the USC Epigenome center and sequenced using 75bp paired end reads on an Illumina HiSeq 2000 according to the manufacturer’s instructions.
RNA-seq was performed after siRNA knockdown using libraries created by GENEWIZ (NJ, USA), and sequenced using 150bp paired-end reads on Illumina HiSeq 4000 by GENEWIZ (NJ, USA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
RNA-seq in A549 cells after MYBL2 siRNA treatment rep4
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| Data processing |
ChIP-seq reads were aligned to the human reference genome hg38 following standards used by the ENCODE3 consortium outlined in Landt SG et al. 2012 and detailed in the ENCODE3 ChIP-seq pipeline (https://www.encodeproject.org/chip-seq/). In brief, bwa v.0.7.17 was used to align the reads, trimming the first 5 base pairs of each read, allowing for two mismatches. Duplicate reads were marked using Picard MarkDuplicates v.2.12.0, and the duplicated reads and reads with mapping quality lower than 30 were removed using samtools v.1.9. Peaks and signal tracks were generated using the Macs2 v.2.1.2 peak caller. For peak calling, the callpeak function was used with a p-value cutoff set to 0.01, no shift selected, an extension size equal to the value calculated by for the ChIP files from cross correlation, with all duplicate tags kept, and a pileup signal file created.
ATAC-seq reads were aligned to the human reference genome hg38 using the same methods performed for ChIP-seq, using the ENCODE3 pipeline for paired end files.
RNA-seq reads were aligned to the human reference genome hg38 using the Genomic Data Commons Bioinformatics mRNA analysis pipeline (https://docs.gdc.cancer.gov/Data/Bioinformatics_Pipelines/Expression_mRNA_Pipeline/). In brief, the two-pass alignment method was performed using STAR v2.6.0a using settings as specified by the pipeline. Read counts were generated for GENCODE v22 genes using the htseq-count function from HTSeq v0.11.1
Genome_build: hg38
Supplementary_files_format_and_content: For ChIP-seq and ATAC-seq, tab-delimited narrowPeak files contain locations of peaks and bigwig files contain read pileup information
Supplementary_files_format_and_content: For RNA-seq, tab-delimited text files contain read counts for GENCODE v22-annotated genes
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| Submission date |
Jan 06, 2020 |
| Last update date |
Aug 21, 2020 |
| Contact name |
Daniel James Mullen |
| E-mail(s) |
dmullen@usc.edu
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| Organization name |
University of Southern California
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| Department |
Biochemistry and Molecular Medicine, Surgery
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| Lab |
Offringa
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| Street address |
1441 Eastlake Ave., Room NTT 6420
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90089 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE143145 |
TENET 2.0: Identification of key transcriptional regulators and enhancers in lung adenocarcinoma |
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| Relations |
| BioSample |
SAMN13738779 |
| SRA |
SRX7503551 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4250952_A549-MYBL2-4_readcounts.txt.gz |
245.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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