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Late pre-B cell transcriptomes from Zfp36l1 Zfp36l2 double knockout mice [iCLIP]

ABSTRACT: Purpose: We aimed to identify the targets of the RNA binding protein ZFP36L1 in B cells. Methods: Mature B cells were stimulated with LPS, IL-4 and IL-5 for 48 hours to induce ZFP36L1 expression. Cells were treated with UV light to crosslink RNA and proteins then RNA-protein complexes were pulled down with anti-ZFP36L1. RNA was extracted and used to make cDNS libraries that were then sequenced using Illumina’s HiSeq2000 (100 bp single end sequencing). Results: Sample demultiplexing was performed by identification of the 3 known bases of the 7 bases barcode introduced in the 5’ end of the read by the RCLIP primer. The remaining four random bases were used to remove PCR duplicate reads. Reads were trimmed to remove any adaptor sequence and barcodes before mapping reads to genome mm10 using Bowtie. After read mapping, the single-nucleotide at position -1 was annotated as unique ZFP36L1 crosslink site. Identification of highly significant ZFP36L1 binding sites was performed using iCount to assign a FDR to each crosslink site Conclusions: We identified ZFP36L1 binding sites in 1361 B cell mRNAs.

ORGANISM(S): Mus musculus

PROVIDER: GSE78239 | GEO | 2016/04/21

SECONDARY ACCESSION(S): PRJNA313059

REPOSITORIES: GEO

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Pre-T cells from control and Zfp36l1, Zfp36l2 double conditional knockout mice. [iCLIP]

Project description:Purpose: We aimed to identify the targets of the RNA binding protein ZFP36L1 in thymoctes. Methods: Total naÃ¯ve thymocytes from control or DCKO mice were treated with UV light to crosslink RNA and proteins, then RNA-protein complexes were pulled down with anti-ZFP36L1. RNA was extracted and used to make cDNS libraries that were then sequenced by MiSeq 150bp single-end read (Sample 1) and HiSeq2500 RapidRun 50bp single-end read (sample 2, 3). Results: Sample demultiplexing was performed by identification of the 3 known bases of the 7 bases barcode introduced in the 5â end of the read by the RCLIP primer. The remaining four random bases were used to remove PCR duplicate reads. Reads were trimmed to remove any adaptor sequence and barcodes before mapping reads to genome GRCm38 using Bowtie. After read mapping, the single-nucleotide at position -1 was annotated as unique ZFP36L1 crosslink site. Identification of highly significant ZFP36L1 binding sites was performed using iCount to assign a FDR to each crosslink site. Conclusions: We identified ZFP36L1 binding sites in 8675 thymocyte mRNAs. Individual nucleotide resolution cross linking immunoprecipitation (iCLIP) of ZFP36L1-bound RNAs in total naive thymocytes from C57BL/6 mice.

2016-09-05 | E-GEOD-79178 | biostudies-arrayexpress

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Project description:Purpose: We aimed to identify the targets of the RNA binding protein ZFP36L1 in thymoctes. Methods: Total naïve thymocytes from control or DCKO mice were treated with UV light to crosslink RNA and proteins, then RNA-protein complexes were pulled down with anti-ZFP36L1. RNA was extracted and used to make cDNS libraries that were then sequenced by MiSeq 150bp single-end read (Sample 1) and HiSeq2500 RapidRun 50bp single-end read (sample 2, 3). Results: Sample demultiplexing was performed by identification of the 3 known bases of the 7 bases barcode introduced in the 5’ end of the read by the RCLIP primer. The remaining four random bases were used to remove PCR duplicate reads. Reads were trimmed to remove any adaptor sequence and barcodes before mapping reads to genome GRCm38 using Bowtie. After read mapping, the single-nucleotide at position -1 was annotated as unique ZFP36L1 crosslink site. Identification of highly significant ZFP36L1 binding sites was performed using iCount to assign a FDR to each crosslink site. Conclusions: We identified ZFP36L1 binding sites in 8675 thymocyte mRNAs.

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2016-04-21 | GSE78240 | GEO

RNAseq of pre-T cells from control and Zfp36l1, Zfp36l2 double conditional knockout mice.

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2016-09-05 | E-GEOD-79174 | biostudies-arrayexpress

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RNAseq of pre-T cells from control and Zfp36l1, Zfp36l2 double conditional knockout mice.

Project description:Purpose: Conditional knockout of Zfp36l1 Zfp36l2 early in lymphocyte development leads to a bypass of beta-selection and subsequently T cell acute lymphoblastic leukemia. This RNA seq experiment aimed to determine the molecular pathways affected by loss of Zfp36l1 and Zfp36l2, and to deduce direct targets of these RNA binding proteins. Methods: RNA was isolated from sorted Zfp36l1fl/fl; Zfp36l2fl/fl DN3a (Lineage-negative, CD44-, Kitlow, CD25+, CD98low) and DN3b (Lineage-negative, CD44-, Kitlow, CD25intermediate, CD98+) cells as well as Zfp36l1fl/fl; Zfp36l2fl/fl; CD2cre DN3 (Lineage-negative, CD44-, Kitlow, CD25+) cells with the RNeasy Micro Kit (Qiagen). RNAseq libraries were prepared from 20-200ng RNA using the TruSeq Stranded Total RNA and rRNA Removal Mix – Gold from Illumina. Libraries were sequenced by Hiseq in 100bp single-end reads. The reads were trimmed to remove adapter sequences using Trim Galore then mapped using Tophat (version 2.0.12) to the GRCm38 mouse assembly; reads with an identical sequence to more than one genomic locus were not mapped. Quality control analysis was carried out with FastQC. Reads were counted using htseq-count tool and mouse gtf file version 78. Results: Differences in the abundance of transcripts between DCKO and control samples were calculated in the R/Bioconductor program DESeq2 (version 1.6.3). Adjusted P values for differential expression were calculated in DESeq2 using a Benjamini-Hochberg correction: genes with an adjusted p-value of less than 5% were considered significant. Differentially expressed mouse transcripts identified using DESeq2 were analyzed for gene set enrichment using Toppfun. Conclusions: We identified an enrichment of mRNAs involved in cell cycle progression within Zfp36l1 Zfp36l2 double conditional knockouts.

2016-09-05 | GSE79174 | GEO

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