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Sample GSM5708162 Query DataSets for GSM5708162
Status Public on Apr 24, 2022
Title Input_2
Sample type SRA
 
Source name B cells
Organism Mus musculus
Characteristics cell type: In vitro LPS-stimulated B cells
mouse strain: C57BL/6
genotype: WT
Growth protocol Splenic B cells were isolated and stimulated for three days in vitro with 5ug/ml LPS
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.
 
Library strategy RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina NextSeq 500
 
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
 
Submission date Nov 29, 2021
Last update date Apr 24, 2022
Contact name Ziv Shulman
E-mail(s) ziv.shulman@weizmann.ac.il
Organization name Weizmann Institute of Science
Department Department of Immunology
Lab Shulman
Street address Herzl St 234
City Rehovot
ZIP/Postal code 76100
Country Israel
 
Platform ID GPL19057
Series (1)
GSE189819 YTHDF2 suppresses the plasmablast genetic program and promotes germinal center formation
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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