(A) Overview of shRNA library design.75 loci associated with red blood cell traits (van der Harst et al., 2012) were used as the basis to calculate 75 genomic windows of LD 0.8 or greater from the …
Table containing annotations and information for the 75 SNPs used to seed the shRNA library.
Table containing annotations and information for all hairpins, as well as shRNA counts for each time point and replicate.
(A) Counts of loci from among the original 75 annotated with linkage to each of the six RBC traits, hemoglobin (Hb), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration …
(A) Schematic of the loss of function lentiviral constructs tested for pooled screens in primary CD34+ cells. (B) FACS plots showing the proportion of infected GFP+ cells 4 days after transduction …
(A) Histogram showing distribution of number of independent hairpins included in the library to target each of the candidate’s genes. (B) Representative FACS plots of erythroid cell surface markers …
(A) Kernel density plot showing library representation as log2 shRNA CPM across all hairpins. (B) shRNA abundance log2 fold changes from day 4 to day 16. Represented values are the mean of hairpin …
Represented values are the mean of hairpin abundance log2 fold changes across hairpins for each gene and two standard deviations.
(A) Bar graph showing the 38 of 75 loci in the screen with at least one corresponding statistically significant (FDR < 0.1, β >0.1) gene effect causing either a positive or negative log2 fold change …
Table containing the R model output for each gene.
(A) Histogram showing the number of gene hits identified at each of the 40 loci with at least one significant gene effect detected. Statistical model output for each gene in screen available in Figur…
(A) Permuted enrichment of essentiality among the set of hit genes vs. randomly chosen sets of genes from the human genome. (B) Permuted enrichment of essentiality among the set of hit genes vs. …
Purple boxes highlight the cell types that were enriched for expression of hit genes.
STRING interaction network analysis identifies signaling/transcription, membrane, and mRNA translation-related subnetworks important to erythropoiesis embedded in the genes identified in the screen …
(A) Quantitative RT-PCR and (B) Western blot showing the expression of TFR2 in human CD34+ cells five days post-infection with the respective lentiviral shRNAs targeting TFR2 (TFR2 sh1 and sh2) and …
(A) Representative FACS plots of alternate erythroid cell surface markers CD49d (α4 integrin) and CD235a (Glycophorin A) expression at various time points during erythroid differentiation. (B) …
(A) Quantitative RT-PCR and (B) Western blot showing the expression of SF3A2 in human CD34+ cells five days post-infection with the respective lentiviral shRNAs targeting SF3A2 (sh1-4) and a control …
Table containing the DESeq2 output for differentially expressed genes in cells undergoing SF3A2 knockdown or control shRNA treatment.
Table containing the DESeq2 output for differentially expressed genes in MDS patients with and without mutations in SF3B1.
Tables containing the GO component (Table 1) and function (Table 2) enrichments calculated using GOrilla for cells undergoing SF3A2 knockdown or control shRNA treatment.
Tables containing the GO component (Table 1) and function (Table 2) enrichments calculated using GOrilla for MDS patient samples with and without mutations in SF3B1.
Tables containing the differential splicing analysis for cells undergoing SF3A2 knockdown or control shRNA treatment.
Categories of splice mutations presented in each table are alternative 3’ splice sites, alternative 5’ splice sites, mutually exclusive exons, retrained introns, and skipped exons, respectively.
Tables containing the differential splicing analysis for MDS patient patient samples with and without mutations in SF3B1.
Categories of splice mutations presented in each table are alternative 3’ splice sites, alternative 5’ splice sites, mutually exclusive exons, retrained introns, and skipped exons, respectively.
(A) shRNAs targeting SF3A2 co-expressing a reporter GFP gene was infected into human CD34+ cells and cultured in erythroid conditions. GFP expression at various time points from three independent …
(A) Knockdown efficiency of shRNAs targeting SF3A2 in murine erythroleukemia (MEL) cells by western blot. (B) Total cell numbers of GFP +shRNA expressing bone marrow cells from wildtype (WT) and Sf3b…
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Biological sample (Homo sapiens) | CD34 + mobilized peripheral blood | Fred Hutchinson Cancer Research Center | ||
Cell line (Homo sapiens) | UT-7/EPO | NA | RRID:CVCL_5202 | maintained in Sankaran laboratory |
Cell line (Mus musculus) | MEL | NA | maintained in Sankaran laboratory | |
Genetic reagent (Mus musculus) | Sf3b1K700E | Obeng et al., 2016 | Dr. Benjamin L. Ebert (Brigham Women's Hospital, Boston MA) | |
Recombinant DNA reagent (lentiviral shRNA) | PLKO.1-Puro (plasmid) | Sigma-Aldrich | RRID :Addgene_10878 | Pol III based shRNA backbone |
Recombinant DNA reagent (lentiviral shRNA) | PLKO-GFP (plasmid) | this paper | GFP version of pLKO.1-Puro | |
Recombinant DNA reagent (lentiviral shRNA) | SFFV-Venus-mir30 shRNA (plasmid) | this paper | Pol II based shRNA backbone | |
Antibody | mouse monoclonal anti-human CD235a-APC | Thermo Fisher Scientific | Cat#: 17-9987-42; RRID:AB_2043823 | FACS (5 ul per test) |
Antibody | mouse monoclonal anti-human CD71-FITC | Thermo Fisher Scientific | Cat#: 11-0719-42; RRID:AB_1724093 | FACS (5 ul per test) |
Antibody | mouse monoclonal anti-human CD71-PEcy7 | Thermo Fisher Scientific | Cat#: 25-0719-42; RRID:AB_2573366 | FACS (5 ul per test) |
Antibody | mouse monoclonal ant-human CD49d-PE | Miltenyi Biotec | Cat#: 130-093-282; RRID:AB_1036224 | FACS (10 ul per test) |
Antibody | mouse monoclonal anti-human CD41a-PE | Thermo Fisher Scientific | Cat#: 12-0419-42; RRID:AB_10870785 | FACS (5 ul per test) |
Antibody | mouse monoclonal anti-human CD11b-PE | Thermo Fisher Scientific | Cat#: 12-0118-42; RRID:AB_2043799 | FACS (5 ul per test) |
Antibody | Rat monoclonal anti-mouse Ter119-APC | Thermo Fisher Scientific | Cat#: 17-5921-82; RRID:AB_469473 | FACS (0.25 ug/test) |
Antibody | Rat monoclonal anti-mouse CD71-PE | Thermo Fisher Scientific | Cat#: 12-0711-82; RRID:AB_465740 | FACS (0.5 ug/test) |
Antibody | mouse monoclonal anti-phospho STAT5 Alexa Fluor-647 | BD Bioscience | Cat#: 612599; RRID:AB_399882 | FACS (1:20) |
Antibody | mouse monoclonal anti-GAPDH | Santa Cruz Biotechnology | sc-32233; RRID:AB_627679 | Western (1:20,000) |
Antibody | mouse monoclonal anti-TFR2 | Santa Cruz Biotechnology | sc-32271; RRID:AB_628395 | Western (1:200) |
Antibody | mouse monoclonal anti-SF3A2 | Santa Cruz Biotechnology | sc-390444 | Western (1:1000) |
Sequence-based reagent | shLUC | Sigma-Aldrich | TRCN0000072259 | 5’- CGCTGAGTACTTCGAAATGTC-3’ |
Sequence-based reagent | TFR2 sh1 (human) | Sigma-Aldrich | TRCN0000063628 | 5’-GCCAGATCACTACGTTGTCAT-3’ |
Sequence-based reagent | TFR2 sh2 (human) | Sigma-Aldrich | TRCN0000063632 | 5-CAACAACATCTTCGGCTGCAT-3’ |
Sequence-based reagent | SF3A2 sh1 (human) | Sigma-Aldrich | TRCN0000000060 | 5’-CTACGAGACCATTGCCTTCAA-3’ |
Sequence-based reagent | SF3A2 sh2 (human) | Sigma-Aldrich | TRCN0000000061 | 5’-CCTGGGCTCCTATGAATGCAA-3’ |
Sequence-based reagent | SF3A2 sh3 (human) | Sigma-Aldrich | TRCN0000000062 | 5’-CAAAGTGACCAAGCAGAGAGA-3’ |
Sequence-based reagent | SF3A2 sh4 (human) | Sigma-Aldrich | TRCN0000000063 | 5’-ACATCAACAAGGACCCGTACT-3’ |
Commercial assay or kit | RNeasy Mini Kit | QIAGEN | Cat#: 74104 | |
Commercial assay or kit | iScript cDNA synthesis Kit | Bio-Rad | Cat#: 1708891 | |
Commercial assay or kit | iQ SYBR Green Supermix | Bio-Rad | Cat#: 170–8882 | |
Commercial assay or kit | NucleoSpin Blood XL-Maxi kit | Clonetch | Cat#: 740950.1 | |
Commercial assay or kit | Lineage Cell Depletion Kit (mouse) | Miltenyi | Cat#: 130-090-858 | |
Commercial assay or kit | Nextera XT DNA Library Preparation Kit | Illumina | Cat#: FC-131–1096 | |
Commercial assay or kit | NextSeq 500/550 High Output Kit v2.5 (75 Cycles) | Illumina | Cat#: 20024906 | |
Commercial assay or kit | Bioanalyzer High Sensitivity DNA Analysis | Agilent | Cat#: 5067–4626 | |
Commercial assay or kit | Agencourt AMPure XP | Beckman-Coulter | Cat#: A63881 | |
Commercial assay or kit | TaKaRa Ex TaqDNA Polymerase | Takara | Cat#: RR001B | |
Commercial assay or kit | Qubit dsDNA HS Assay Kit | Thermo Fisher | Cat#: Q32854 | |
Chemical compound, drug | Human Holo-Transferrin | Sigma Aldrich | Cat#: T0665-1G | |
Peptide, recombinant protein | Humulin R (insulin) | Lilly | NDC 0002-8215-01 | |
Peptide, recombinant protein | Heparin | Hospira | NDC 00409-2720-01 | |
Peptide, recombinant protein | Epogen (recombinant erythropoietin) | Amgen | NDC 55513-267-10 | |
Peptide, recombinant protein | Recombinant human stem cell factor (SCF) | Peprotech | Cat#: 300–07 | |
Peptide, recombinant protein | Recombinant human interleukin-3 (IL-3) | Peprotech | Cat#: 200–03 | |
Peptide, recombinant protein | Recombinant mousestem cell factor (SCF) | R&D systems | Cat# 455-MC-010 | |
Peptide, recombinant protein | recombinant mouse Insulin like Growth Factor 1 (IGF1) | R&D systems | Cat# 791 MG-050 | |
Chemical compound, drug | Hoechst 33342 | Life Technologies | Cat#: H1399 | FACS (1:1000) |
Chemical compound, drug | Fixation Buffer | BD Bioscience | Cat#: 554655 | |
Chemical compound, drug | Perm Buffer III | BD Bioscience | Cat#: 558050 | |
Chemical compound, drug | May-Grünwald Stain | Sigma-Aldrich | Cat#: MG500 | |
Chemical compound, drug | Giemsa Stain | Sigma-Aldrich | Cat#: GS500 | |
Software, algorithm | STAR | Dobin et al., 2013 | RRID:SCR_015899 | |
Software, algorithm | MISO | Katz et al., 2010 | RRID:SCR_003124 | |
Software, algorithm | R | The R Foundation | RRID:SCR_001905 | |
Software, algorithm | Salmon | Patro et al., 2017 | RRID:SCR_017036 | |
Software, algorithm | GOrilla | Eden et al., 2009 | RRID:SCR_006848 | |
Software, algorithm | VEP | McLaren et al., 2016 | RRID:SCR_007931 | |
Software, algorithm | FlowJo version 10 | FlowJo | RRID:SCR_008520 | |
Software, algorithm | GraphPad Prism 7 | GraphPad Software Inc | RRID:SCR_002798 | |
Software, algorithm | Python 2, 3 | Python Software Foundation | RRID:SCR_008394 | |
Software, algorithm | PLINK | Chang et al., 2015 | RRID:SCR_001757 | |
Software, algorithm | PoolQ | Broad Institute | https://portals.broadinstitute.org/gpp/public/software/poolq |