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GSE166404

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

Organism: Homo sapiens
Platform: GPL28352
Samples: 2
Experiment Types:
Other
Submitted: Feb 08 2021
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/PRJNA700773 SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRP305418

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 yields RBP target sites in an isoform-specific manner. Finally, Ribo-STAMP leverages small ribosomal subunits to measure transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.

Overall Design

strategy for immunoprecipitation-free detection of protein-RNA interactions by fusion of RNA binding proteins and ribosomal subunits to the C-to-U RNA editing enzyme APOBEC1 and quantification of interaction-driven edits by sequencing, to allow interaction detection on long-read and single-cell platforms

Analysis (9 steps)

View Data Processing
Processing steps for GSE166404
  1. The double stranded cDNA for each sample was converted to sequencing libraries as recommended (PacBio SMRTbell Express Template Prep Kit 2.0) but with separate barcoded adapters for each sample (PacBio Barcoded Overhang Adapter Kit).
  2. All of the samples were pooled in an equimolar fashion and sequenced on a SMRTcell 8M with the PacBio Sequel II instrument (2.0 chemistry/2.1 polymerase with 2 hour pre-extension and 30 hour movie times).
  3. After barcodes were demultiplexed, the initial data was used to re-balance the pooling by barcode counts before further sequencing.
  4. In total, the samples were sequenced over 5 SMRTcell 8M.
  5. The PacBio Sequel II system was used for all sample sequencing.
  6. Following sequencing, the circular consensus sequence (CCS) reads for each set of technical replicates were processed using the Isoseq v3 pipeline (https://github.com/PacificBiosciences/IsoSeq) to generate full-length non-concatamer reads in fasta format.
  7. For this step, software package lima v2.0.0 was used with parameters: --isoseq and --dump-clips.
  8. In addition, isoseq3 v3.4.0 refine was used with parameters: --require-polya.
Showing first 8 steps.

Supplementary Files (1)

GSE166404_RAW.tar Download
GEO Samples (2)
GSM5070701 GSM5070702

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 (2) and Runs (30)

Total: 2413 MB
SRX10055416 SRP305418 OTHER SINGLE
GSM5070701: HEK293T, RBFOX; Homo sapiens; OTHER
Sample: SRS8218002
BioProject: PRJNA700773
BioSample: SAMN17838657
Platform: PACBIO_SMRT
Instrument: Sequel II
Organism: Homo sapiens
Sample attributes
source_name: Human embryonic kidney
cell line: HEK293T
Original files (1)
Human embryonic kidney
Runs (15)
Run Spots Bases Size (MB) Files Link
SRR13663731 7819 23584116 12.59 CCS_Demux_5759_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc1020_BAK8B_OA.b… SRA
SRR13663732 33920 88891256 38.77 CCS_Demux_6465_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc1020_BAK8B_OA.b… SRA
SRR13663733 40240 111682035 49.16 CCS_Demux_6465_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc1021_BAK8B_OA.b… SRA
SRR13663734 41468 119623016 52.88 CCS_Demux_6465_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc1022_BAK8B_OA.b… SRA
SRR13663735 14122 36417519 17.35 CCS_Demux_6499_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc1020_BAK8B_OA.b… SRA
SRR13663736 20757 57142553 27.18 CCS_Demux_6499_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc1021_BAK8B_OA.b… SRA
SRR13663737 28329 81454972 39.11 CCS_Demux_6499_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc1022_BAK8B_OA.b… SRA
SRR13663738 11134 36092902 19.52 CCS_Demux_5759_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc1021_BAK8B_OA.b… SRA
SRR13663739 15576 51926719 28.24 CCS_Demux_5759_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc1022_BAK8B_OA.b… SRA
SRR13663740 126649 312535539 141.21 CCS_Demux_5779_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc1020_BAK8B_OA.b… SRA
SRR13663741 172903 458184347 208.61 CCS_Demux_5779_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc1021_BAK8B_OA.b… SRA
SRR13663742 227556 635178702 293.33 CCS_Demux_5779_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc1022_BAK8B_OA.b… SRA
SRR13663743 25297 73575652 39.63 CCS_Demux_5896_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc1020_BAK8B_OA.b… SRA
SRR13663744 33417 102691880 55.5 CCS_Demux_5896_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc1021_BAK8B_OA.b… SRA
SRR13663745 34567 110242738 60.42 CCS_Demux_5896_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc1022_BAK8B_OA.b… SRA
SRX10055417 SRP305418 OTHER SINGLE
GSM5070702: HEK293T, APOBEC; Homo sapiens; OTHER
Sample: SRS8218003
BioProject: PRJNA700773
BioSample: SAMN17838656
Platform: PACBIO_SMRT
Instrument: Sequel II
Organism: Homo sapiens
Sample attributes
source_name: Human embryonic kidney
cell line: HEK293T
Original files (1)
Human embryonic kidney
Runs (15)
Run Spots Bases Size (MB) Files Link
SRR13663746 17645 59069914 32.04 CCS_Demux_5759_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1001_BAK8A_OA.ba… SRA
SRR13663747 30042 84841382 37.64 CCS_Demux_6465_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1001_BAK8A_OA.ba… SRA
SRR13663748 40988 123788148 55.67 CCS_Demux_6465_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1015_BAK8B_OA.ba… SRA
SRR13663749 32342 85159180 37.04 CCS_Demux_6465_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1016_BAK8B_OA.ba… SRA
SRR13663750 31519 90700478 43.78 CCS_Demux_6499_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1001_BAK8A_OA.ba… SRA
SRR13663751 23807 69995865 34.3 CCS_Demux_6499_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1015_BAK8B_OA.ba… SRA
SRR13663752 22347 60755233 28.82 CCS_Demux_6499_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1016_BAK8B_OA.ba… SRA
SRR13663753 15574 54256116 29.66 CCS_Demux_5759_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1015_BAK8B_OA.ba… SRA
SRR13663754 12289 38824781 20.71 CCS_Demux_5759_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1016_BAK8B_OA.ba… SRA
SRR13663755 251917 710473192 327.6 CCS_Demux_5779_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1001_BAK8A_OA.ba… SRA
SRR13663756 216623 627311943 291.68 CCS_Demux_5779_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1015_BAK8B_OA.ba… SRA
SRR13663757 194939 498775739 228.51 CCS_Demux_5779_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1016_BAK8B_OA.ba… SRA
SRR13663758 30285 95829587 52.67 CCS_Demux_5896_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1001_BAK8A_OA.ba… SRA
SRR13663759 34268 114452662 63.04 CCS_Demux_5896_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1015_BAK8B_OA.ba… SRA
SRR13663760 29034 86070839 46.54 CCS_Demux_5896_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1016_BAK8B_OA.ba… 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 (30)

Accession File Name Stored Type Output Type Mapping Assembly Size Download
— CCS_Demux_5759_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1… OTHER 32.0 MB link
— CCS_Demux_5759_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1… OTHER 29.7 MB link
— CCS_Demux_5759_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1… OTHER 20.7 MB link
— CCS_Demux_5759_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc… OTHER 12.6 MB link
— CCS_Demux_5759_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc… OTHER 19.5 MB link
— CCS_Demux_5759_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc… OTHER 28.2 MB link
— CCS_Demux_5779_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1… OTHER 327.6 MB link
— CCS_Demux_5779_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1… OTHER 291.7 MB link
— CCS_Demux_5779_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1… OTHER 228.5 MB link
— CCS_Demux_5779_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc… OTHER 141.2 MB link
— CCS_Demux_5779_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc… OTHER 208.6 MB link
— CCS_Demux_5779_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc… OTHER 293.3 MB link
— CCS_Demux_5896_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1… OTHER 52.7 MB link
— CCS_Demux_5896_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1… OTHER 63.0 MB link
— CCS_Demux_5896_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1… OTHER 46.5 MB link
— CCS_Demux_5896_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc… OTHER 39.6 MB link
— CCS_Demux_5896_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc… OTHER 55.5 MB link
— CCS_Demux_5896_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc… OTHER 60.4 MB link
— CCS_Demux_6465_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1… OTHER 37.6 MB link
— CCS_Demux_6465_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1… OTHER 55.7 MB link
— CCS_Demux_6465_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1… OTHER 37.0 MB link
— CCS_Demux_6465_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc… OTHER 38.8 MB link
— CCS_Demux_6465_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc… OTHER 49.2 MB link
— CCS_Demux_6465_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc… OTHER 52.9 MB link
— CCS_Demux_6499_APO1_1_demultiplex.bc1001_BAK8A_OA__bc1… OTHER 43.8 MB link
— CCS_Demux_6499_APO1_2_demultiplex.bc1015_BAK8B_OA__bc1… OTHER 34.3 MB link
— CCS_Demux_6499_APO1_3_demultiplex.bc1016_BAK8B_OA__bc1… OTHER 28.8 MB link
— CCS_Demux_6499_RBFOX_1_demultiplex.bc1020_BAK8B_OA__bc… OTHER 17.3 MB link
— CCS_Demux_6499_RBFOX_2_demultiplex.bc1021_BAK8B_OA__bc… OTHER 27.2 MB link
— CCS_Demux_6499_RBFOX_3_demultiplex.bc1022_BAK8B_OA__bc… OTHER 39.1 MB link