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GEO help: Mouse over screen elements for information. |
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Status |
Public on Apr 26, 2024 |
Title |
BMDM csRNA-seq notx SPRET rep2 |
Sample type |
SRA |
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Source name |
TSS mapping in untreated BMDMs from SPRET mice (rep2)
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Organism |
Mus musculus |
Characteristics |
cell type: Primary bone marrow derived macrophages assay: csRNA-seq (transcription initiation) genetic perturbation: none treatment: none mouse strain: SPRET
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Growth protocol |
U2OS cells were grown at 37°C in DMEM (Cellgro) supplemented with 10% FBS (Gibco), 50 U Penicillin and 50 μg Streptomycin per mL (Gibco). For siRNA transfection, cells were washed once with PBS (Gibco), trypsinized for about 5 minutes and then washed twice with PBS (Gibco) by centrifugation at 400 g for 5 minutes. Subsequently, about 3 million cells were resuspended in 150 µl Gene Pulser® Electroporation Buffer (Biorad) and siRNAs in 1x siRNA buffer (60 mM KCl, 6 mM Hepes 7.5, 0.2 mM MgCl2). The following siRNAs were used: M-011796-02- 0005 siGENOME Human YY1 (7528) siRNA, M-017924-01- 0005 siGENOME Human NRF1 (4899) siRNA, and siGFP (CCACUACCUGAGCACCCAGU, (Meade et al. 2014)) as control. For transfection of the dominant-negative NRF1 mutant (AA1-304 (Gugneja, Virbasius, and Scarpulla 1996)), sequence was cloned into pGEM-T and mRNA synthesized using the mMESSAGE mMACHINE™ T7 Transcription Kit (Thermo Fisher Scientific). siRNAs and dnNRF1 were used at a final concentration of 10 μM. Mixture was transferred to a 0.2 cm cuvette and pulsed once at 250 V for a constant 20 msec. Following electroporation, 1 ml complete growth media was added and cells grown for 24h in a 6 cm dish. Bone marrow derived macrophages (BMDMs) from SPRET mice were generated and treated with KLA as described in Link et. al (2018) [PMID:29779944]. TSS-MPRA experiments in HEK293T cells: MPRA inserts sequences were ordered as 200 bp oligos from Twist Bioscience and cloned Gibson-assembled into a BsaI-linearized pTSS-MPRA-Empty plasmid based on the background-reduced pGL4.10 reporter backbone (Promega), with a cloning site harboring the 18-bp pMPRA1-LH sequence followed by tandem BsaI sites for linearization and the TruSeq Read 2-compatible pMPRA1-RH sequence and a downstream landing site for reverse transcription primer RS2, and an EGFP ORF replacing the Luciferase 2 gene. About 800k HEK293T cells were used and 25 µg plasmid DNA was transfected using Lipofectamine® 3000 (Thermo Fisher Scientific) as described by the manufacturer. After 8 h, cells were washed 3x with PBS (Gibco) and RNA extracted using 1 ml Trizol (Thermo Fisher Scientific).
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Extracted molecule |
total RNA |
Extraction protocol |
For all csRNA, RNA-seq, and TSS-MPRA experiments, to harvest RNA, plates were washed three times using ice cold PBS and 1 ml TRIzol™ Reagent added. Cells were scraped and RNA extracted as described by the manufacturer with addition of 1 μl 15 mg/ml GlycoBlue™ Coprecipitant (Thermo Fisher Scientific). For ChIP-seq, cells were fixed for 10 minutes with 1% formaldehyde/PBS, and the extracted chromatin was sheared to an average DNA size of 300–500 bp. csRNA-seq was performed as described in (Duttke et al. 2019). Small RNAs of ~20-60 nt were size selected from 0.4-2 µg of total RNA by denaturing gel electrophoresis. A 10% input sample was taken aside and the remainder enriched for 5’-capped RNAs. Monophosphorylated RNAs were selectively degraded by 1 hour incubation with Terminator 5´-Phosphate-Dependent Exonuclease (Lucigen). Subsequently, RNAs were 5’dephosporylated through 90 minutes incubation in total with thermostable QuickCIP (NEB) in which the samples were briefly heated to 75°C and quickly chilled on ice at the 60 minutes mark. small RNA input (csRNAinput-seq) and csRNA-seq libraries were prepared as described in (Hetzel et al. 2016) using RppH (NEB) and the NEBNext Small RNA Library Prep kit, amplified for 14 cycles and sequenced SE75 on the Illumina NextSeq 500. Strand-specific total RNA-seq libraries from ribosomal RNA-depleted RNA were prepared using the TruSeq kit Stranded Total RNA Library kit (Illumina) and sequenced PE100 on Illumina NovaSeq 6000. For ChIP-seq, one percent of the lysate was kept as ChIP input, and dnNFR1-HA was immunoprecipiated with 2 μg anti-HA antibody (Abcam ab9110) and 20 μl of Dynabeads Protein A. Libraries were prepared directly on the antibody/chromatin-bound beads using NEBNext Ultra II reagents according to the manufacturer’s protocol. Libraries were sequenced single-end for 75 cycles (SE75) to a depth of 20-25 million reads on an Illumina NextSeq 500 instrument. For TSS-MPRA, starting with 10-15 µg RNA (⅓ of the total), non-capped RNAs were dephosphorylated with Quick CIP (NEB) and DNA digested. Samples were mixed well and incubated at 37°C for 90 minutes. For more complete dephosphorylation of uncapped transcripts, RNA was denatured a second time at 75°C for 30 seconds in a pre-warmed water bath and then quickly chilled on ice for 2 minutes before incubating the sample at 37°C for another 30 minutes. As Quick CIP is thermolabile, the brief heat treatment leaves the enzyme active. Following Cap-enrichment, RNA was purified and then vortexed hard and subsequently spun for 10 minutes at 12k g at RT. Following phase separation, the upper layer was taken, and RNA precipitated. RNA pellets were then resuspended and 5' caps removed. Following decapping, 5’ adapters were ligated by T4RNA Ligase 1 and RNA extracted. RNA pellets were then resuspended and reverse transcribed. Samples were amplified and sequenced paired-end on a NextSeq 500 (additional details available in the manuscript)
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Description |
short RNAs (20-65nt) tss.BMDM.SpretVsC57Bl6.notx.bed.gz
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Data processing |
csRNA-seq (small capped RNAs, ~20-60 nt) and csRNAinput-seq reads were trimmed of their adapter sequences using HOMER (“homerTools trim -3 AGATCGGAAGAGCACACGTCT -mis 2 -minMatchLength 4 -min 20”). Reads for all genome-based assays (i.e. everything except TSS-MPRA) were aligned to the appropriate genome (GRCh38/hg38, GRCm38/mm10, ChlSab1.1) using STAR version 2.7.9a with default parameters. Only reads with a single, unique alignment (MAPQ >=10) were considered in the downstream analysis. In the case of mouse SPRET BMDM csRNA-seq, these data were analyzed in the context of C57Bl6 BMDM csRNA-seq previously published as part of GSE135498 [PMID:31649059]. To compare how TSS change between the two strains of mice, only TSS positions that were homologous between C57Bl6 and SPRET genomes and were uniquely alignable in both genomes were considered for downstream analysis (additional details available in the manuscript) To analyze TSS positions, csRNA/csRNAinput-seq reads with spliced or soft clipped alignments were first discarded to ensure accurate TSS assignments. The alignment position of the 5’ nucleotide of each csRNA-seq read was used to create a map of putative TSS locations in the genome for each sample set. To ensure we use high quality TSS that could be reliably quantified across different conditions, only TSS locations with at least 7 reads per 10^7 total aligned reads across all compared replicates and conditions (e.g. Ctrl siRNA, NRF1 siRNA, etc.) were kept for further analysis. Furthermore, any TSS position that had higher normalized read density in the small csRNAinput-seq data was discarded as a likely false positive TSS location. These sites often include miRNAs and other high abundance RNA species which are not entirely depleted in the csRNA-seq cap enrichment protocol. To quantify the change in TSS levels between conditions, a unified map of confident TSS positions is first determined across the set of compared experiment as described above. Then, the TSS activity levels are assessed for each replicate and each experimental condition by first counting the raw read coverage and normalizing the dataset using DESeq2’s rlog variance stabilizing transform [PMID: 25516281]. Changes in transcriptional activity were then reported as the log2 fold change representing the difference between averaged rlog transformed activity levels across conditions (similar to a shrunken fold change). ChIP-seq peaks were found using HOMER's findPeaks program using "-style factor". TSS-MPRA anlysis: Raw sequencing reads were trimmed for the 5’ adapter sequence GGTAACCGGTCCAGCTCA on the R1 read using cutadapt version 3.4. The trimmed reads were aligned to the reporter library using STAR version 2.7.9a, specifying an option to preclude soft-clipping at the 5’ end of R1 (STAR --outSAMattributes All --genomeDir library/tfsweep.STARIndex --runThreadN 12 --readFilesIn file.fastq --outFileNamePrefix star/Sweep_1. --alignEndsType Extend5pOfRead1 --outSAMtype BAM SortedByCoordinate). For DNA plasmid control samples, the uniquely aligned read pairs were counted to later scale transcriptional output. For RNA samples, uniquely aligned read pairs were further processed to identify exact transcription start sites, yielding a matrix of start sites per sequence position. Any alignments showing mismatches at their 5’ ends were not counted and reporter sequences with fewer than 50 total DNA alignments were also ignored. A scaling factor for each sequence was determined by calculating min(10000/plasmid, 100). After multiplying the start site counts by the scaling factor, a pseudocount of 1 was added and values were log2 transformed. Assembly: hg38, mm10, ChlSab1.1 Supplementary files format and content: bed files: Locations indicate TSS or ChIP-seq peak positions. Numerical values associated with the files represent normalized read densities or log2 ratios between two conditions as indicated by the file names. fa files: FASTA files containing the designed insert sequences used for TSS-MPRA experiments tsv files: tab-delimited text files containing the TSS activity levels for each TSS-MPRA insert. Each column represents the TSS position starting within the insert starting from the beginning of the variable sequence region. fpkm.txt file: tab-delimited text file containing RNA-seq FPKM expression values for GENCODE annotated genes.
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Submission date |
Mar 25, 2022 |
Last update date |
Apr 26, 2024 |
Contact name |
Christopher Benner |
E-mail(s) |
cbenner@ucsd.edu
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Organization name |
University of California, San Diego (UCSD)
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Department |
Medicine
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Street address |
9500 Gilman Dr. MC 0640
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City |
La Jolla |
State/province |
California |
ZIP/Postal code |
92093-0640 |
Country |
USA |
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Platform ID |
GPL19057 |
Series (1) |
GSE199431 |
Position-dependent function of human sequence-specific transcription factors |
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Relations |
BioSample |
SAMN26957679 |
SRA |
SRX14617585 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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