|
| Status |
Public on Dec 06, 2023 |
| Title |
KARR-seq F123 ribo-depletion (-) Rep 1 |
| Sample type |
SRA |
| |
|
| Source name |
KARR-seq F123 ribo-depletion (-)
|
| Organism |
Mus musculus |
| Characteristics |
cell line: F123
cell type: embryonic stem cells
treatment: None
|
| Treatment protocol |
Cells were crosslinked in 1% formaldehyde for 10 min and then quenched with 125 mM glycine for 5 min. Cells were incubated 30 min in lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA-630, 5 mM EDTA) supplemented with 20 mM N3-kethoxal, proteinase inhibitor, and RNase inhibitor, and was collected by centrifuging. The cell pellet was then washed and shook in lysis buffer supplemented with 12.5 µM G1-DBCO-biotin dendrimer at 1,000 rpm at 37 ˚C for 1 h. After the reaction, cells were collected and washed once as above.
|
| Growth protocol |
HEK293T and HepG2 cells were cultured in DMEM (Gibco 11995) supplemented with 10% (v/v) fetal bovine serum (Gibco), 1% penicillin and streptomycin (Gibco) at 37 ˚C with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Resuspend cell pellets in 410 µL 25 mM K3BO3, with 50 µL 10% SDS, 30 µL proteinase K, 10 µL RNase inhibitor. The mixture was shaken at 55 ˚C for 2 h and was subjected to phenol-chloroform extraction and ethanol precipitation. Extracted RNA were used for enrichment, end repair, and proximity ligation.
Libraries were conctructed by using the SMARTer Stranded Total RNA-seq kit v2 – pico input mammalian.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
G1_kethoxal-riboplus_B06_R01
|
| Data processing |
library strategy: KARR-seq
KARR-seq: For paired end FASTQ formatted files, SeqPrep (commit: 575507b) was first used to merge overlapping read pairs (2 x 150 bp) into single end reads. Unmerged read pairs were discarded. The resulting single end results were then aligned to mm10, hg19 or dm3 reference genome using STAR (v2.5.2a) as well as the species' corresponding RefSeq transcripts. The pre-merging step was skipped for single-end FASTQ files, and directly aligned using STAR. The following STAR parameters were used to recover gapped and chimeric reads (--runMode alignReads --outFilterMultimapNmax 100 --outSAMattributes all --alignIntronMin 1 --scoreGapNoncan -4 --scoreGapATAC -4 --chimSegmentMin 15 --chimJunctionOverhangMin 15 --limitOutSJcollapsed 10000000 --limitIObufferSize 1500000000). Only chimeric reads (CIGAR N gap between mapped segments of the read) were kept, spliced sites filtered and interaction pairs tabulated. Pairix was used to index the interaction pairs file.
Genome_build: mm10,hg19,dm3
Supplementary_files_format_and_content: Processed files are bgzipped text files that follows the 4DN-standard pairs file format (pairix)
|
| |
|
| Submission date |
Feb 04, 2021 |
| Last update date |
Dec 06, 2023 |
| Contact name |
Youzhi Cheng |
| Organization name |
University of Connecticut Health Center
|
| Department |
Genetics and Genome Sciences
|
| Street address |
400 Farmington Avenue
|
| City |
Farmington |
| State/province |
Connecticut |
| ZIP/Postal code |
06032 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE166155 |
KARR-seq reveals principles of high-order ribonucleoprotein structure assembly |
|
| Relations |
| BioSample |
SAMN17792692 |
| SRA |
SRX10021308 |
