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GSE155726

GSE GEO
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Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [scRNA-seq]

Organism: Homo sapiens
Platform: GPL24676
Samples: 5
Experiment Types:
Expression profiling by high throughput sequencing
Submitted: Aug 05 2020
Last Updated: Jan 24 2025
Status: Public on May 09 2021
Contact: Gene,,Yeo (UCSD)

Relations

SubSeries of: GSE155729 BioProject: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA655413 SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRP276054

Summary

RNA binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet, the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC Mediated Profiling), which efficiently detects RBP RNA interactions. STAMP does not rely on UV crosslinking or immunoprecipitation and, when coupled with single cell capture, can identify RBP and cell type specific RNA protein interactions for multiple RBPs and cell types in single pooled experiments. Pairing STAMP with long read sequencing also yields RBP target sites for full length isoforms. Finally, conducting STAMP using small ribosomal subunits (RiboSTAMP) allows analysis of transcriptome wide ribosome association in single cells. STAMP enables the study of RBP RNA interactomes and translational landscapes with unprecedented cellular resolution.

Overall Design

Cells expressing RBP-APOBEC1 fusions for C-to-U editing of RBP target-RNAs were processed through the Chromium Single Cell Gene Expression Solution using the Chromium Single Cell 3’ Gel Bead, Chip, 3’ Library and 3’ Feature Barcode Library Kits v3 (10X Genomics) as per the manufacturer’s protocol. The following fusion expressions were processed for a target of 10,000 cells/ experiment: transient transfection of control- and RBFOX2-APOBEC1 (STAMP) in HEK293T and separately in neural progenitor cells (NPC), independent stable inductions of control- and RBFOX2:TIA1-STAMP (cell mixture, capture sequence barcoded), and RPS2-STAMP.

Analysis (12 steps)

View Data Processing
Processing steps for GSE155726
  1. Alignment, cell barcode processing, umis processing, abundance measurements: cellranger count (version 3.0.2)
  2. MD tags were added to alignments with samtools calmd --threads 15 -rb possorted_genome_bam.bam refdata-cellranger-hg19-3.0.0/fasta/genome.fa > possorted_genome_bam_MD.bam
  3. Reads were split based on the CB:Z tag, resulting in one BAM file per barcode.
  4. Edits were called on each "split-BAM" file using SAILOR (http://github.com/yeolab/sailor) using default parameters and dbSNP (v147) to remove known SNPs.
  5. Coverage was generated from filtered BAM files (intermediates from SAILOR) for each cell barcode, using the following commands from BedTools (v2.27.1). :
  6. bedtools genomecov -split -strand - -g refdata-cellranger-hg19-3.0.0/star/chrNameLength.txt -bg -ibam fwd.bam > fwd.bg
  7. bedtools sort -I fwd.bg > fwd.sorted.bg
  8. bedGraphToBigWig fwd.sorted.bg refdata-cellranger-hg19-3.0.0/star/chrNameLength.txt fwd.sorted.bw
Showing first 8 steps.

Supplementary Files (1)

GSE155726_RAW.tar Download
GEO Samples (5)
GSM4711280 GSM4711281 GSM4711282 GSM4711283 GSM4711284

Dataset Citations (2)

Robust single-cell discovery of RNA targets of RNA-binding proteins and ribosomes.
PMID 33963355 · 2021 · Nature methods
Kristopher W Brannan, Isaac A Chaim, Ryan J Marina, Brian A Yee, Eric R Kofman, Daniel A Lorenz, Pratibha Jagannatha, Kevin D Dong, Assael A Madrigal, Jason G Underwood, Gene W Yeo
Long-read Ribo-STAMP simultaneously measures transcription and translation with isoform resolution.
PMID 38906680 · 2024 · Genome research
Pratibha Jagannatha, Alexandra T Tankka, Daniel A Lorenz, Tao Yu, Brian A Yee, Kristopher W Brannan, Cathy J Zhou, Jason G Underwood, Gene W Yeo

SRA Experiments (5) and Runs (5)

Total: 61531 MB
SRX8889932 SRP276054 RNA-Seq PAIRED
GSM4711280: NPC_HEK_control-STAMP; Homo sapiens; RNA-Seq
Sample: SRS7149411
BioProject: PRJNA655413
BioSample: SAMN15734793
Platform: ILLUMINA
Instrument: Illumina NovaSeq 6000
Organism: Homo sapiens
Sample attributes
source_name: HEK293T/NPC mix
cell line: HEK293T/NPC mix
expressing: APOBEC1
Original files (1)
HEK293T/NPC mix
Runs (1)
Run Spots Bases Size (MB) Files Link
SRR12391497 444501891 43561185318 8849.38 NPC_HEK_control_STAMP_possorted_genome_bam.bam, SRR12391497, SRR12391… SRA
SRX8889933 SRP276054 RNA-Seq PAIRED
GSM4711281: NPC_HEK_RBFOX2-STAMP; Homo sapiens; RNA-Seq
Sample: SRS7149412
BioProject: PRJNA655413
BioSample: SAMN15734791
Platform: ILLUMINA
Instrument: Illumina NovaSeq 6000
Organism: Homo sapiens
Sample attributes
source_name: HEK293T/NPC mix
cell line: HEK293T/NPC mix
expressing: RBFOX2
Original files (1)
HEK293T/NPC mix
Runs (1)
Run Spots Bases Size (MB) Files Link
SRR12391498 465313539 45600726822 8816.15 NPC_HEK_RBFOX2_STAMP_possorted_genome_bam.bam, SRR12391498, SRR123914… SRA
SRX8889934 SRP276054 RNA-Seq PAIRED
GSM4711282: RBP-STAMP; Homo sapiens; RNA-Seq
Sample: SRS7149413
BioProject: PRJNA655413
BioSample: SAMN15734789
Platform: ILLUMINA
Instrument: Illumina NovaSeq 6000
Organism: Homo sapiens
Sample attributes
source_name: HEK293T
cell line: HEK293T
expressing: RBFOX2/TIA1
Original files (1)
HEK293T
Runs (1)
Run Spots Bases Size (MB) Files Link
SRR12391499 887636932 81107291842 19043.22 RBP_STAMP_possorted_genome_bam.bam, SRR12391499, SRR12391499.lite SRA
SRX8889935 SRP276054 RNA-Seq PAIRED
GSM4711283: control-STAMP; Homo sapiens; RNA-Seq
Sample: SRS7149414
BioProject: PRJNA655413
BioSample: SAMN15734788
Platform: ILLUMINA
Instrument: Illumina NovaSeq 6000
Organism: Homo sapiens
Sample attributes
source_name: HEK293T
cell line: HEK293T
expressing: APOBEC1
Original files (1)
HEK293T
Runs (1)
Run Spots Bases Size (MB) Files Link
SRR12391500 569302766 55791671068 12260.94 control_STAMP_possorted_genome_bam.bam, SRR12391500, SRR12391500.lite SRA
SRX8889936 SRP276054 RNA-Seq PAIRED
GSM4711284: RPS2-STAMP; Homo sapiens; RNA-Seq
Sample: SRS7149415
BioProject: PRJNA655413
BioSample: SAMN15734787
Platform: ILLUMINA
Instrument: Illumina NovaSeq 6000
Organism: Homo sapiens
Sample attributes
source_name: HEK293T
cell line: HEK293T
expressing: RPS2
Original files (1)
HEK293T
Runs (1)
Run Spots Bases Size (MB) Files Link
SRR12391501 587033102 57529243996 12561.09 RPS2_STAMP_possorted_genome_bam.bam, SRR12391501, SRR12391501.lite SRA

Linked Publications (3)

Long-read Ribo-STAMP simultaneously measures transcription and translation with isoform resolution.
Genome research · 2024
Robust single-cell discovery of RNA targets of RNA-binding proteins and ribosomes.
Nature methods · 2021
FLARE: a fast and flexible workflow for identifying RNA editing foci.
BMC bioinformatics · 2023

Data Files (20)

Accession File Name Stored Type Output Type Mapping Assembly Size Download
— control_STAMP_possorted_genome_bam.bam RNA-Seq 12.0 GB link
— control_STAMP_possorted_genome_bam.bam RNA-Seq 12.0 GB link
— control_STAMP_possorted_genome_bam.bam RNA-Seq 12.0 GB link
— control_STAMP_possorted_genome_bam.bam RNA-Seq 12.0 GB link
— NPC_HEK_control_STAMP_possorted_genome_bam.bam RNA-Seq 8.6 GB link
— NPC_HEK_control_STAMP_possorted_genome_bam.bam RNA-Seq 8.6 GB link
— NPC_HEK_control_STAMP_possorted_genome_bam.bam RNA-Seq 8.6 GB link
— NPC_HEK_control_STAMP_possorted_genome_bam.bam RNA-Seq 8.6 GB link
— NPC_HEK_RBFOX2_STAMP_possorted_genome_bam.bam RNA-Seq 8.6 GB link
— NPC_HEK_RBFOX2_STAMP_possorted_genome_bam.bam RNA-Seq 8.6 GB link
— NPC_HEK_RBFOX2_STAMP_possorted_genome_bam.bam RNA-Seq 8.6 GB link
— NPC_HEK_RBFOX2_STAMP_possorted_genome_bam.bam RNA-Seq 8.6 GB link
— RBP_STAMP_possorted_genome_bam.bam RNA-Seq 18.6 GB link
— RBP_STAMP_possorted_genome_bam.bam RNA-Seq 18.6 GB link
— RBP_STAMP_possorted_genome_bam.bam RNA-Seq 18.6 GB link
— RBP_STAMP_possorted_genome_bam.bam RNA-Seq 18.6 GB link
— RPS2_STAMP_possorted_genome_bam.bam RNA-Seq 12.3 GB link
— RPS2_STAMP_possorted_genome_bam.bam RNA-Seq 12.3 GB link
— RPS2_STAMP_possorted_genome_bam.bam RNA-Seq 12.3 GB link
— RPS2_STAMP_possorted_genome_bam.bam RNA-Seq 12.3 GB link