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Blood, Vol. 113, Issue 6, 1340-1349, February 5, 2009
Mir-144 selectively regulates embryonic  -hemoglobin synthesis during primitive erythropoiesis
Blood Fu et al.
113: 1340
Supplemental materials for: Fu et al
Embryonic chromatin immunoprecipitation (E-ChiP)
For each immunoprecipitation, 130 embryos at 16 hpf and 100 embryos at 22 hpf were enzymatically dechorionated and then fixed in 1.85% formaldehyde in 1× embryo medium for 20 minutes at room temperature. Glycine (0.125 M) was added to quench the formaldehyde, and the embryos were homogenized in swelling buffer (25 mM Hepes, pH 7.8, 1.5 mM MgCl2, 10 mM KCl, 0.5% NP-40, 1 mM DTT, and Protease inhibitors), and incubated for 20 minutes on ice. Nuclei were collected by centrifugation, resuspended in nuclei lysis buffer (20 mM pH 8.0-Tris–HCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 0.1% Na-deoxycholate, 150 mM NaCl, and protease inhibitors), then incubated for 10 minutes and sonicated on ice. Sonication conditions were optimized to give fragments of approximately 300 to 700 bp. The lysates were pre-cleared by constant rotation with salmon sperm DNA/Protein-A beads (Upstate) (use 40 µl Sepharose per ml lysate) for 1 hour at 4°C, then centrifuge samples at 8000 rpm for 1 minute. The supernatant was incubated with 5 µg of rabbit anti-Klfd antibody, then rotated overnight at 4°C. 30 µl salmon sperm DNA/Protein-A beads per IP (equilibrated as above) were added and rotated 1 hour at 4°C. The beads were centrifuged at 4°C and washed with washing buffer (as described in the ChiP assay kit protocol, Upstate Biotechnology). Bound complexes were eluted from the beads at RT with vortexing 10 minutes in elution buffer. Cross-links were reversed for 4 hours at 65°C, and the chromatins were digested with RNase A, followed by proteinase K treatment and phenol:chloroform:isoamyl alcohol extraction. The ChiP experiments were carried out four times with separate batches of embryos. PCR products were separated with a 1.5% agarose gel. Primer sequences were listed in Table S3.
Files in this Data Supplement:
- Table S1. Stable transgenic lines established in this study (PDF, 70.8 KB)
- Table S2. Target prediction of miR-144 for genes expressed in Intermediate Cell Mass (ICM) (PDF, 134 KB)
- Table S3. Primers sequences used in this study (PDF, 113 KB)
- Figure S1. Expression of zebrafish miR-144 during embryogenesis (JPG, 73.6 KB)
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Whole-mount miRNA in situ hybridization was performed at the indicated stages with a Dig-labeled, LNA-modified oligonucleotides as a probe. Embryos are lateral views with the animal pole up (A–F, 0.75–12 hpf), and head to the left, dorsal upwards (G–L, 18–120 hpf). The eye is indicated (e) as a point of reference.
- Figure S2. Efficient suppression of EGFP reporter expression by miR-144 LNA (JPG, 102 KB)
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(A,B) Co-injection of EGFP-2×PT sensor mRNAs with either miR-144 duplex control (A) or miR-144 duplex (B) into wild-type embryos. Fluorescent steromicroscope shows EGFP expression at 22 hpf. The bright-field images of corresponding embryos are shown below. (C) Co-injection of miR-144 duplex with either EGFP-2×PT or EGFP-2×PTmut sensor mRNAs. Fluorescent steromicroscope shows EGFP expression at 22 hpf. (D,E) WISH analysis using Dig-labeled EGFP antisense mRNAs as a probe in the 22 hpf embryos injected with either EGFP-2×PT (D) or EGFP-2×PTmut (E) sensor mRNAs. (F,G) Co-injection of EGFP-2×PT sensor mRNAs and miR-144 duplex with either miR-144 LNA control (F) or miR-144 LNA (G) into wild-type embryos. Fluorescent steromicroscope shows EGFP expression at 22 hpf. The bright-field images of corresponding embryos are shown below. (H) Northern blot analysis of mature miR-144 and miR-1 expression in 22 hpf embryos injected with 0.3 ng of either miR-144 LNA or miR-144 LNA control.
- Figure S3. Representative results of α-E1 globin expression (JPG, 62.8 KB)
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Embryos injected with either miR-144 LNA control (left), and miR-144 LNA (right). Arrows denote embryos with massive increase of α-E1 globin expression.
- Figure S4. Mature erythroid gene expressions in miR-144 knockdown embryos (JPG, 108 KB)
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(A–D) WISH analysis of hemoglobins α-E1, α-E3, β-E1, and β-E2 expressions in the control- and miR-144 LNA-injected embryos at 22, 28, 33, and 48 hpf. The symbols “♥” denote images that have been used in Figure 3.
- Figure S5. Erythroid progenitor and hematopoietic stem cell gene expressions in miR-144 knockdown embryos (JPG, 103 KB)
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(A–D) WISH analysis of alas2, gata-1, fog 1, and scl expressions in the control- and miR-144 LNA-injected embryos at 22, 28, 33, and 48 hpf. The symbols “♥” denote images that have been used in Figure 3.
- Figure S6. Granulocytic and vascular gene expressions in miR-144 knockdown embryos (JPG, 102 KB)
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(A–D) WISH analysis of pu.1, mpo, l-plastin, and flk 1 expressions in the control- and miR-144 LNA-injected embryos at 22, 28, 33, and 48 hpf. The symbols “♥” denote images that have been used in Figure 3.
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