Team:BM-AMU/Protocols

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Protocols

Plasmid construction

PCR

PCR primers

ECad

hECad-LA：

hECad-LXF: 5’- CGTTCTAGAAACTTGCTTGGTTAC -3’

XbaI: TCTAGA；

hECad-LBR: 5’- CAAGGATCCGTCGTCCTCGCCGC -3’

BamHI: GGATCC

hECad-RA：

hECad-REF: 5’- TTGGATATCTAGGGGACTCGAG -3’

EcoRV (GAT/ATC)

hECad-RKR: 5’- CAAGGTACCAAGATGTGGCCAGAC -3’

Kpn I (GGTAC/C)

NCad

hNCad-LA：

hNCad-LF: 5’- GGGCTGCAGGAATTCGATTGGCAGGCTTGATCTCTCAAG -3’

hNCad-LR: 5’- GTCATCACCTCCACCATACATGTC -3’

hNCad-RA：

hNCad-RF: 5’- GATATCTGAACTTCAGGGTGAACTTG -3’

hNCad-RR: 5’- ACGGTATCGATAAGCTTGATGATGGGAAGGTCCAAAATGA -3’

（ATCAAGCTTATCGATACCGT - pBKSII）

EGFP (773 bp, for Gibson assembly)

hNCad-GFP-F: 5’- ATGGTGGAGGTGATGACGGAGGAGGAGGAATGGTGA -3’

hNCad-GFP-R: 5’- CCCTGAAGTTCAGATATCTCACGTACGCTTGTA -3’

(GAGCTGTACAAGCGTACGTGAGATATCTGAACTTCAGGGTGAACTTG – GFP-hNCadherin)

PCR amplification system

Template                          4μL

Upstream primers            2μL

Downstream primers       2μL

dNTP                               10μL

MgSO4                             7.5μL

Buffer                               10μL

KOD-PLUS                        2μL

H2O                                 62.5μL

TOTAL                              100μL

PCR amplification system

94℃,3min;

[98℃,15sec;60℃,30sec;68℃,2.5min]（×30）;

68℃,5min.

Enzyme digestion system

H2O (30μL) + Plasmid skeleton (20μL) + Buffer(6μL) + enzyme 1(2μL) + enzyme 2(2μL) Total = 60μL → 37°C，3-5hrs .

Gel extraction

According to manufacturer's instructions.

Ligation

system: Vector(0.3μL)+insert(1.2μL)+2*Quick ligase buffer(1.5μL)+T4 Ligase(0.2μL) =3.2μL.

condition: 25℃，20min，Incubation in PCR.

Transformation

a)DH5α competent cells melt immediately on ice.

b)condition：25℃，20min，Incubation in PCR.

c) 42.5 C water bath heat shock for 1 minute and 15 seconds, quickly placed on ice for 2 minutes.

d) Prepare the LB medium in advance. Bacterial culture tube was taken, antibiotic-free LB medium was taken by suction tube and added into the shaker at 37 ℃, 200 r, 30 min for resuscitation.

e) Prepare a formula containing antibiotics. Mix the above bacterial . solution and coat it with 100 mL. Apply the L-shaped bar evenly until it absorbs completely.

f) Inverted plate was incubated at 37 ℃ for overnight.

g) Selection of bacteria（The next day）.

h) Prepare the culture tube in advance and add the ampicillin-containing medium in two pipettes.

i) Working in a super-clean platform, two separate and symmetrical colonies were selected and mixed in the culture tube.

j) The culture tube was placed in a shaker at 37℃ for 6 (12) - 16 h (no more than 24 h).

Plasmid Extraction

According to manufacturer's instructions.

Cell transfection

We use Lipo 2000 for cell transfection

According to manufacturer's instructions.

We also used LONZA 4D for cell transfection

According to manufacturer's instructions.

Fluorescence knock-in identification

PCR

KOD FX system 20μL

Buffer                     10μL

dNTPs                     4μL

primer F                  0.5μL

primer R                 0.5μL

DNA                      1μL

H2O                      3.5μL

KOD FX                  0.5μL

Condition：94℃，2min-98℃,10s-60℃,30s-68℃(1000bp/min)-72℃,5min

RNA extraction

According to manufacturer's instructions.

Western blot

FACS Analyses and Sorting. HMLER cells were trypsinized and tumors dissociated using a tumor isolation kit (Milteny). One million single cells in sus- pensions per 100 μL were stained with appropriate antibody dilution (SI Appendix, SI Materials and Methods) in 2% IFS/PBS for 15–30 min RT in the dark. DAPI was used for live–dead analysis. Cells were directly resuspended in PBS and analyzed on a LSRII flow and sorted on a FACS Aria (BD Biosciences). Data were analyzed using the FlowJo software (Tree Star). To purify the CD104+ and CD104− populations the top 5% of the positive and negative spectrum were isolated.

Construction of monitor

Evaluation of WA09 Cell Line Steady Transferred by TetO-SNAIL1-TWIST-ERT2

DOX+TetO-SNAIL1

a) 12-well plate, cell 3X10^5/well; the experimental biology was repeated twice.

b) DOX concentration gradient: 0 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml.

c) The expression time of Tet-off in the reference manual was 9h-RμL=3X105, which could be observed 6 hours after the addition of the drug. Photographic recording of tomato intensity changes.

d) Flow test preparation, PBS cleaning, digestion and centrifugation followed by 500-1000 ml 30% glucose suspension.

e) Western Blot was used to detect the expression trend of ECAD, NCAD and SNAIL1.

Tamoxifen+TWIST1-ERT2

a) 12-well plate, cell 3X10^5/hole. The experimental biology was repeated three times.

b) Tamoxifen concentration gradient: 0μl/ml，2μl/ml，4μl/ml，6μl/ml，8μl/ml，10μl/ml.

c) The changes of tomato intensity were observed frequently and recorded by photography.

d) Flow test preparation, PBS cleaning, digestion and centrifugation followed by 500-1000 mL 30% glucose suspension .

e) Identification of relative contents of ECAD, NCAD and TWIST by qRT-PCR.

Attention: If the dose-effect relationship is not good, the time-effect relationship experiment can be carried out. The concentration of DOX is 1 μg/ml and that of Tamoxifen is 5 μg/ml

CAG Promoter Efficiency Measurement Experiment

Aim: To compare the expression of GFP (flow cytometry) in 293 cells by promoter CAG, CMV, EF1a and Ubiquitin.

Cells: 24-well plate, 2X105/hole, four groups of experiments each set up two biological repeats. 3X106 cells (10cm plate) are needed.

Transfection

A. 800ng plasmid +100μLOpti-mem.

B. 2μL lipo2000+100μLOpti-mem (The same system can be configured together).

Mix at room temperature for 5 minutes, and mix A and B at room temperature for 20 minutes. The transfection was observed 24 hours later.

Comparing the efficiency of rtTA, TetR and TetR-KRAB

a) Construction of TetO-GFP cell line in 293-FT cells.

b) 3-5 X105 cell suspension infection virus was inoculated into 6-well plate and cultured for 1-2 days. Cells were screened by neomycin 250ug/ml.

c) Cells：24-well plate, 2X105/hole, three groups of experiments each set up two biological repeats. 2X106 cells (10cm plate) were needed. The cells in the 6-well plate could be divided into three holes in the 24-well plate and cultured to 90% adherent transfection or suspension transfection after digestion.

d) lipo2000 transfection( 24-well culture plate ):

A. 800ng rtTA +100μLOpti-mem

B. 800ng TetR+100μLOpti-mem

C. 800ng TetR-KRAB+100μLOpti-mem

D. 3*2=6μL lipo2000+300μLOpti-mem

e) Mix at room temperature for 5 minutes, A. B. C and 100 μLD. Mix at room temperature for 20 minutes.

f) Add into Dox: After 24 hours, one is divided into two to 24 orifices.

g) The first line was set as control, and the second line was added with DOX (1 ug/ml). After 24 hours of cμLture, the relative fluorescence expression was recorded by photography and detected by flow cytometry.

RNA-Seq Sample Preparation

RNA-Seq. Q-PCR was performed as described before (19) (SI Appendix, Sup- plementary Materials and Methods). RNA-Seq libraries were prepared using the TruSeq-stranded polA mRNA kits (Illumina). Libraries were pooled and sequenced on the HiSeq 2500 sequencer, 40 bp reads to a depth of ∼40– 45 million mapped. RNA-Seq data from this study have been deposited at GEO under accession number GSE119149. RNA-Seq paired-end reads from Illumina 1.5 encoding were aligned using TopHat (v.2.0.12) (52) to the hu- man genome (GRCh37) with Ensemble annotation (GRCh37.75) in gtf format. Differentially expressed genes were hierarchical clustered using uncentered.