Supplementary Data

Antibodies

Primary antibodies and dilutions are indicated as followed. For immunofluorescent staining; mouse Anti-Nestin (1:200) (Chemicon, Cat. no. MAB5326), rabbit Anti-Sox2 (1:200) (Chemicon, Cat. no. MAB4343), rabbit Anti-TUJ1 (1:500) (BioLegend, Cat. no. MRB-435P), rabbit Anti-GFAP (1:500) (Millipore, Cat.no.AB5804), mouse Anti-Olig2 (1:500) (Chemicon, Cat. no. MABN50), rabbit Anti-Netrin-1 (1:200) (Abcam, Cat. no. EPR5428), FITC conjugated- mouse Anti-von Willebrand Factor (vWF) (1:500) (R&D Systems, Cat. no. FAB932F) and PE-conjugated mouse Anti-CD146 (1:500) (BioLegend, Cat. no. 342004). For Flow cytometry; PE-conjugated Anti-CD133/1 (Miltenyi Biotec, Cat. no. 130-080-801), PE-conjugated Anti-CD34 (BioLegend, Cat. no. 343606), PerCP-conjugated Anti-CD45 (BioLegend, Cat. no. 304026), FITC conjugated- mouse Anti-vWF and PE-conjugated mouse Anti-CD146. For western blot; rabbit Anti-Netrin-1 (1:1000) and mouse anti-b-actin (1:1000) (Cell signaling, Cat. no. 3700). Secondary antibodies and dilutions are indicated as followed. For immunofluorescent staining; Alexa Fluor® 488 goat anti-mouse (1:500) (Invitrogen, Cat. no. 1IVM-A11001), Alexa Fluor® 568 goat anti-mouse (1:500) (Invitrogen, Cat. no. 1IVM-A11004), Alexa Fluor 488® goat anti-rabbit (1:500) (Invitrogen, Cat. no. 1IVM-A11008) and Alexa Fluor® 568 goat anti-rabbit (1:500) (Invitrogen, Cat. no. 1IVM-A11011). For western blot; HRP- conjugated goat Anti-rabbit IgG (1:5000) (Millipore, Cat. no.12-348) and HRP- conjugated goat Anti-mouse IgG (1:5000) (Novagen, Cat. no. 71045-3).

Plasmid construction

Plasmid construction protocol was indicated as 4 main steps as following:

1. Generating ds-oligos of pre-Netrin-1 miR sequences

Single stranded-oligonucleotides (ss-oligo) of pre-Netrin-1 miR sequences (top and bottom) were purchased from Invitrogen. Each lyophilized ss-oligos was resuspended in TE buffer to make a final concentration of 200 μM. Reaction tubes were set up as following; 5 μl of top strand ss-oligo (200 μM), 5 μl of bottom strand ss-oligo (200 μM), 2 μl of 10x Oligo annealing buffer and 8 ml of DNase/RNase-free water. For annealing reaction, tubes were incubated at 95°C for 4 minutes, and allowed the mixture to cool to 25oC for 10 minutes in thermal cycler machine. One microliter of the annealed mixture (50 μM ds-oligo) was taken out for preparing dilution. The remainders were stored at -20°C.

2. Creating entry clones

The ds-oligos from previous step were ligated into linearized pcDNA™6.2-GW/ EmGFP miR vector (5,699 bp) to create pcDNA™6.2-GW/EmGFP pre-Netrin-1 miR RNAi expression vector (5,759 bp) by performing ligation reaction. The ligation tubes were set up as following: 4 μl of 5x ligation buffer, 2 μl of linearized pcDNA™6.2/GW-EmGFP-miR (5 ng/μl), 4 μl of each Netrin-1 miR ds-oligo (10 nM), 9 μl of DNase/RNase-free water and 1 μl of T4 DNA ligase (1 U/μl). Ligation reaction tubes were incubated at 25oC for 15 minutes, and placed on ice until performing TOP10 competent E.coli transformation or stored at -20oC until use.

For TOP10 competent E.coli transformation, 2 µl of each ligation product was added into a vial of TOP10 competent E.coli and mixed gently. Tubes were incubated on ice for 15 minutes and performed heat-shock in 42°C water bath for 30 seconds without shaking. Afterthat, tubes were immediately transferred on ice. SOC medium at 250 µl was added into each tube and incubated with shaking (200 rpm) at 37°C for 1 hour. After incubation, transformed bacteria at particular volumes, 50 ml and 200 ml, were spread onto each LB agar plate containing 50 μg/ml spectinomycin. Another 100 ml was spread onto a LB agar plates without antibiotics (control plate).

Three spectinomycin-resistant colonies were picked, and then grew in 5 ml LB broth with 50 μg/ml spectinomycin at 37oC for 16-18 hours on horizontal shakers. Each bacterial suspension was taken to purify for transformant plasmid DNA (pcDNA™6.2-GW/EmGFP-pre-Netrin-1 miR vector) by Midiprep kit (Geneaid) according to the manufacturer’s instruction. Purified plasmid DNA was measured DNA concentration by spectrophotometer at 260 nm. Afterthat each transformant was analyzed by PCR and verified by DNA sequencing analysis to confirm the insert sequence (pre-Netrin-1 miR ds-oligo).

PCR was performed for 40 cycles using EmGFP forward primer and miRNA reverse primer. Sequences of primers are indicated as in Table S2. PCR cycle was shown as following: initial denaturation at 95oC for 10 minutes, followed by 40 cycles of denaturation at 95oC for 30 seconds, annealing at 60oC for 40 seconds and extension at 72oC for 60 seconds, and final elongation at 72oC for 5 minutes and hold the reaction at 4oC or kept in -20oC until analysis by gel electrophoresis. PCR product of each transformants was shown at 274 bp.

For DNA sequencing, purified plasmid DNA was prepared in nuclease-free water at 100 ng/ml for 50 ml. Samples were analyzed by outsource DNA sequencing service using EmGFP forward primer and miRNA reverse primer. Sequences of primers are indicated as in Table S3. DNA sequencing results were then aligned with known input sequences using online bioinformatics’ tool from EMBOSS Matcher nucleotide alignment which should be shown more than 95% matching for overall sequences and must be 100% matching at ds-oligo insertion part of the plasmid.

3. BP recombination

BP recombination reaction (attB x attP = attL) was performed using Gateway® BP Clonase II enzyme to create attL-containing BP recombinants (3,491 bp) from attB-containing pcDNA™6.2-GW/EmGFP pre-Netrin-1 miR RNAi expression vector (from previous step) (5,759 bp) and attP-containing pDONOR™221 vector (4,761 bp). BP reaction tubes were set up as following: 1-7 ml of attB-containing pre-Netrin1 miR expression clones (50 fmol), 1 ml of pDONOR™221 vector (150 ng/ml), TE buffer pH 8.0 to make a volume of 8 ml and 2 ml of BP Clonase II enzyme. Reaction tubes were mixed and incubated at 25oC for 18 hours. After incubation, 1 ml of Proteinase K solution was added into each tube and performed TOP10 competent E.coli transformation.

For TOP10 competent E.coli transformation, 1 ml of BP recombinant was added into a vial of TOP10 competent E.coli. Tubes were incubated on ice for 30 minutes and performed heat-shock in 42°C water bath for 30 seconds without shaking. Afterthat, tubes were immediately transferred on ice for 2 minutes. SOC medium at 250 µl was added into each tube and incubated with shaking (200 rpm) at 37°C for 1 hour. After incubation, transformed bacteria were diluted with LB broth to a volume of 1,000 ml. Transformed cells at 200 ml and 700 ml were spread onto two LB agar plates containing 50 μg/ml kanamycin. Another 100 ml was spread onto LB agar plates without antibiotics (as a control plate). Plates were left for air dry for 10 minutes before incubating overnight at 37°C.

Three kanamycin-resistant colonies were picked, and then grew in 5 ml LB broth with 50 μg/ml kanamycin by shaking at 37oC for 16-18 hours. Each bacterial suspension was taken to purify for BP recombinant plasmid DNA by Midiprep kit (Geneaid). Purified plasmid DNA was measured DNA concentration by spectrophotometer at 260 nm. Afterthat, each BP recombinant was analyzed by PCR and PvuI Restriction enzyme digestion.

PCR was performed for 40 cycles using M13 forward primer and M13 reverse primer. Sequences of primers are indicated as in Table S2. PCR reaction set up and PCR cycle was mentioned earlier. PCR products was kept at -20oC until analysis by gel electrophoresis which should be shown at 1,236 bp.

For restriction enzyme digestion using PvuI, reaction tubes were set up as following: 16-16.5 ml of nuclease-free water, 2 ml of 10x recommended buffer, 1 ml of purified BP recombinants DNA (1 mg), and 1 ml of PvuI restriction enzyme (1U). Reaction tube was mixed gently and incubated at 37oC for 1-16 hours. After incubation, digestion products were analyzed by gel electrophoresis. The product size should be shown at 891 and 2,600 bp.

4. LR recombination

LR recombination reaction (attL x attR = attB) was performed by using Gateway® LR Clonase II enzyme to create LR recombinants or pLenti6/V5-EmGFP-Netrin-1 miR RNAi expression vector (7,979 bp) from attL-containing pre-Netrin1 miR expression plasmids (from BP recombinant) and attR-containing pLenti6/V5-DEST vector (8,688 bp). LR reaction tubes were set up as following: 1-7 ml of attL-containing pre-Netrin1 miR expression plasmid (150 ng/reaction), 1 ml of pLenti6/V5-DEST vector (150 ng/ml), and TE buffer pH 8 to make a volume of 8 ml and 2 ml LR Clonase II enzyme. Reaction tubes were mixed and incubated at 25oC for 1 hour. After incubation, 1 ml of Proteinase K solution was added into each tube and performed Stbl3™ competent E.coli transformation.

For Stbl3™ competent E.coli transformation, 2 ml of LR recombinant was added into a vial of Stbl3™ competent E.coli. Tubes were incubated on ice for 30 minutes and performed heat-shock in 42°C water bath for 45 seconds without shaking. Afterthat, tubes were immediately transferred on ice for 2 minutes. SOC medium at 250 µl was added into each tube and incubated with shaking (225 rpm) at 37°C for 1 hour. After incubation, transformed bacteria were diluted with LB broth to a volume of 1,000 ml. Transformed cells at 200 ml and 700 ml were spread onto two LB agar plates containing 100 μg/ml ampicillin. Another 100 ml was spread onto LB agar plates without antibiotics (as a control plate). Plates were left for air dry for 10 minutes before incubating overnight at 37°C.

Three ampicillin-resistant colonies were picked, and then grew in 5 ml LB broth with 100 μg/ml ampicillin by shaking at 37oC for 16-18 hours. Each bacterial suspension was taken to purify for BP recombinant plasmid DNA by Midiprep kit (Geneaid). Each purified plasmid DNA was measured DNA concentration by spectrophotometer at 260 nm. Afterthat, each LR recombinant was analyzed and verified by PCR and DNA sequencing analysis.

PCR was performed for 40 cycles using CMV forward primer and V5 (C-term) reverse primer. Sequences of primers are indicated as in Table S2. PCR reaction set up and PCR cycle was mentioned earlier. PCR products was kept at -20oC until analysis by gel electrophoresis which should be shown at 1,245 bp.

For DNA sequencing, purified plasmid DNA was prepared in nuclease-free water at 100 ng/ml for 50 ml. Samples were analyzed by outsource DNA sequencing service using CMV forward primer and V5 (C-term) reverse primer. Sequences of primers are indicated as in Table S3. DNA sequencing results were then aligned with known input sequences using online bioinformatics’ tool from EMBOSS Matcher nucleotide alignment which should be shown more than 95% matching for overall sequences and must be 100% matching at ds-oligo insertion part of the plasmid.