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Status |
Public on Apr 24, 2022 |
Title |
Input_2 |
Sample type |
SRA |
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Source name |
B cells
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Organism |
Mus musculus |
Characteristics |
cell type: In vitro LPS-stimulated B cells mouse strain: C57BL/6 genotype: WT
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Growth protocol |
Splenic B cells were isolated and stimulated for three days in vitro with 5ug/ml LPS
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Extracted molecule |
polyA RNA |
Extraction protocol |
Total RNA was harvested from activated cells using Trizol. Enrichment for PolyA RNA was done twice using mRNA mRNA direct kit and checked using TapeStation (Agilent). PolyA-selected RNA was barcoded and immunoprecipitated in two rounds using anti-m6A polyclonal antibody bound to magnetic protein-G beads and protein-A beads Libraries were prepared as previously described (Garcia-Campos et al. Cell, 2019). Briefly, eluted RNA was reverse transcribed and an adapter was added using Superscript III reverse transcriptase (Thermo Fisher, 18080093). A second adaptor was added to the cDNA by ligation with T4 RNA Ligase 1 (NEB, M0437M). Following clean-up with MyOne Silane beads, the cDNA library was amplified in a PCR using KAPA HiFi HotStart ReadyMix (KAPA Biosystems KK2601).Libraries were sequenced on an Illumina NextSeq 500 machine.
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Library strategy |
RIP-Seq |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina NextSeq 500 |
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Data processing |
Mars-seq: Reads were trimmed using Cutadapt Mars-seq and m6A-IP: Reads were aligned to the genome using STAR Mars-seq: Quantification was done using htseq-count as previously described in R. Kohen, BMC Bioinformatics, 2019. m6A-IP: Normalization of paired libraries insert size was performed using in-house python scripts, between pairs of INPUT-IP samples. Alignment sorting and indexing were performed separately with samtools (V. 1.3.1) (Li et al. Bioinformatics, 2009). m6A-IP: Identification of putative m6A sites was performed by assigning peak over input (POI) and peak over median (POM) scores to each site, as previously published (Schwartz et al, Cell 2014) single-cell RNAseq: FASTQ files were aligned to the mouse mm10 reference genome using 10x Genomics Cell Ranger software v5.0.1 single-cell RNAseq: The UMI count matrix was converted to Seurat objects using R package Seurat single-cell RNAseq: SCTransform function was used to normalize each dataset Genome_build: Mars-seq, single-cell RNAseq: mm10, m6A-IP: mm9 Supplementary_files_format_and_content: MARS-seq: Excel file with Deseq2 output Supplementary_files_format_and_content: m6A-IP: bam.tdf
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Submission date |
Nov 29, 2021 |
Last update date |
Apr 24, 2022 |
Contact name |
Ziv Shulman |
E-mail(s) |
ziv.shulman@weizmann.ac.il
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Organization name |
Weizmann Institute of Science
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Department |
Department of Immunology
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Lab |
Shulman
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Street address |
Herzl St 234
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City |
Rehovot |
ZIP/Postal code |
76100 |
Country |
Israel |
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Platform ID |
GPL19057 |
Series (1) |
GSE189819 |
YTHDF2 suppresses the plasmablast genetic program and promotes germinal center formation |
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Relations |
BioSample |
SAMN23492981 |
SRA |
SRX13255624 |
Supplementary file |
Size |
Download |
File type/resource |
GSM5708162_Input_2.bam.tdf |
100.8 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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