GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5708148

Query DataSets for GSM5708148

Status

Public on Apr 24, 2022

Title

Exp1_Ythdf2_cKO_3

Sample type

SRA

Source name

B cells

Organism

Mus musculus

Characteristics

cell type: Activated B cells
mouse strain: C57BL/6
genotype: B1-8hi CD23cre Ythdf2fl/fl Tomato+

Treatment protocol

Splenic B cells were adoptively transferred to WT mice, which were immunized with NP-KLH for five days. FAS+ GL-7+ cells were isolated by cell sorting.

Extracted molecule

polyA RNA

Extraction protocol

mRNA was extracted from sorted cells using mRNA direct kit (Invitrogen) and mRNA from each sample was barcoded during reverse transcription and pooled.
Libraries were prepared using a bulk adaptation of the previously described protocol (Keren-Shaul et al. Nat. Protoc, 2019 and Jaitin et al. Science, 2014).Following Agencourt Ampure XP bead cleanup (Beckman Coulter), the pooled samples underwent second strand synthesis and were linearly amplified by T7 in vitro transcription. The resulting RNA was fragmented and converted into a sequencing-ready library by tagging the samples with Illumina sequences during ligation, RT, and PCR. Libraries were quantified by Qubit and TapeStation as well as by qPCR for the ACTB gene as previously described. Sequencing was done using a Nextseq 75 cycle high output kit (Illumina; paired end sequencing).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

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