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Series GSE125118 Query DataSets for GSE125118
Status Public on Aug 31, 2021
Title Mutant U2AF1-Induced Alternative Splicing of H2afy (macroH2A1) Regulates B-Lymphopoiesis in Mice
Organism Mus musculus
Experiment type Expression profiling by high throughput sequencing
Summary Acquired spliceosome gene mutations are among the most common genetic alterations in myelodysplastic syndromes (MDS). Here we present evidence that H2AFY(macroH2A1), a histone H2A variant, is a functional target that is alternatively spliced by mutant U2AF1(S34F), a spliceosome gene. Expression of H2AFY1.1, a H2AFY splice-isoform that is reduced by U2AF1(S34F) expression, rescues the reduction in B-cells observed in U2AF1(S34F) mice. Human MDS samples with U2AF1 mutations have a similar reduction in B-cells. Collectively, our data suggest that altered splicing of H2AFY contributes to MDS pathogenesis.
 
Overall design B-cell biased-lymphoid progenitors were isolated from H2afy+/+ and H2afy-/-mice for RNA-seq. First, bone marrow cells were enriched for c-Kit+ cells using Cd117 MicroBeads on the autoMACS Pro Separator (Miltenyi Biotec). Subsequently, cells were stained according to the staining scheme described above, and BLPs were sorted by FACS (Moflo, Beckman Coulter) directly into the lysis buffer of NucleoSpin RNA XS kit (MACHEREY-NAGEL). RNA was isolated per manufacturer’s instructions. Per sample, RNA equivalent of 5000-15000 BLPs from 2-3 mice were pooled. Total RNA integrity and concentration were assessed using a 2100 Bioanalyzer and an RNA 6000 Pico kit (Agilent). Double stranded-cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per manufacturer’s protocol. cDNA was fragmented using a Covaris E220 sonicator using duty cycle 10, intensity 5, cycles/burst 200, time 180 seconds. Fragments were sequenced on Illumina HiSeq-3000 using single reads extending 50 bases. Reads were aligned to mouse mm9 reference genome using Hisat2(version2.0.6) and following analyses were prefomed using DESeq2 in R
 
Contributor(s) Kim S, Srivatsan SN, Walter MJ
Citation(s) 34469727
Submission date Jan 15, 2019
Last update date Sep 29, 2021
Contact name Matthew J Walter
E-mail(s) mjwalter@wustl.edu
Organization name Washington University School of Medicine
Department Oncology
Lab Southwest Tower, 613L
Street address 660 South Euclid Avenue
City St Louis
State/province MISSOURI
ZIP/Postal code 63011
Country USA
 
Platforms (1)
GPL21493 Illumina HiSeq 3000 (Mus musculus)
Samples (8)
GSM3563767 KO_1
GSM3563768 KO_2
GSM3563769 KO_3
Relations
BioProject PRJNA515274
SRA SRP179618

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Supplementary file Size Download File type/resource
GSE125118_mergedCounts.tsv.gz 379.7 Kb (ftp)(http) TSV
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Raw data are available in SRA
Processed data are available on Series record

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