Functional HOXA13 was removed from BAR-T cells using CRISPR/Cas9-mediated gene editing. A HOXA13 sgRNA targeting exon 1 was cloned into pTLCV2, by ligating two annealed oligonucleotides, i.e. Guide1sgRNA F and R (Supplementary Table 4 ). TLCV2 was a gift from Adam Karpf (Addgene #87360)72 (link). Following sequence verification, the pTLCV2-HOXA13sgRNA plasmid was packaged into lentiviral particles by cotransfection into HEK293T cells with pSPAX2 and pMD2.G, gifts from Didier Trono (Addgene #12260 and #12259). The supernatant was harvested and ultracentrifuged after which BAR-T cells were transduced. Mixed populations of transduced cells were plated at very low confluence, single cell clones could subsequently be isolated using glass cloning cylinders and low melting point agarose from Sigma-Aldrich, followed by DNA isolation using the Kleargene kit, followed by sequence verification with primers TILHOXA13R3 and Pre-HOXA13-FW2 flanking the sgRNA-site (Supplementary Table 4 ). Three cell lines in which both alleles were affected by unique out-of-frame deletions were selected along with three control cell lines.
Glass cloning cylinders
Manufactured by Merck Group
Glass cloning cylinders are transparent, cylindrical containers used in laboratory settings. They are designed to isolate and contain small samples or specimens during experiments or processes. The core function of these cylinders is to provide a controlled and protected environment for the materials within.
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Lab products found in correlation
Low melting point agarose, by Merck Group (1 mentions)
Trypsin edta 1x, by Thermo Fisher Scientific (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Fugene 6, by Promega (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Pspcas9 bb 2a puro px459 v2, by Addgene (1 mentions)
Tet on system, by Takara Bio (1 mentions)
6 protocols using glass cloning cylinders
1
CRISPR-Mediated HOXA13 Gene Knockout
2
Reprogramming Factors Optimize iPSC Generation
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F1 hybrid MEFs at P2 were plated and expanded until reaching confluency of 40% per well in a gelatin-coated 6 well plate and cultured in KSR, FBS, KSR/FBS or FBS+VitC culture conditions (see below) with 1.5 µg/ml of DOX (Cat# D9891, Sigma-Aldrich). The medium was changed every 48 h. Individual iPSCs colonies were picked at day 12 after DOX induction using glass cloning cylinders (Sigma-Aldrich) and 0.05% Trypsin-EDTA 1X (Cat# 25300-054, ThermoFisher Scientific) and plated into a previously gelatin-coated 96-well plate on feeders without DOX. Each iPSC colony was then transferred subsequently into 24, 12 and 6-well plates and 25 cm2 culture flasks. Each iPSC was then continuously passed for 2–4 passages until reaching approximately day 50 after initiation of reprogramming. All cells and reprogramming experiments were performed in an incubator at 37 °C and 5% CO2 in normoxia conditions. The same batch of female and male MEFs derived from the single embryos of the same progeny were used to generate KSR-iPSCs and FBS-iPSCs. Another batch of male hybrid MEFs from another progeny were used to generate KSR/FBS-iPSCs and FBS+VitC-iPSCs.
3
Establishing Stable HEK293 Cell Lines
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Human embryonic kidney (HEK)293 cells were cultured on matrigel-coated plasticware. HEK293 or SH-SY5Y cells grown in Dulbecco's modified Eagle's medium (DMEM), 10% fetal calf serum supplemented with glutamine were transfected in 6 cm diameter culture dishes using DNA mixed with Fugene 6 (Promega) in Optimem I (Invitrogen) and multiple distinct clones for each construct were isolated using glass cloning cylinders (Sigma–Aldrich) following selection with G418. On average, eight clones for each construct were screened for response to TRP channel agonists.
4
Inducible KIF20A Knockdown in Daoy Cells
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Daoy cells were infected with lentivirus of Tet-on-inducible human KIF20A shRNA-726 and GFP-Luciferase prepared from Tet-pLKO-shKIF20A726 plasmid and Hiv7CMV-GFP-IRES-Luciferase plasmid, respectively. The transduced Daoy cells were selected for stable single clones with Puromycin (1 μg/ml, Sigma-Aldrich) and GFP fluorescence signal in DMEM (Invitrogen) with 10% fetal bovine serum. Single clones were picked up by glass cloning cylinders (Sigma-Aldrich) in 10 cm plate. Three clones were obtained and tested for growth ability and doxycycline (0.5 μg/ml) inducibility using growth curve assay, western blottings, and luciferase reporter assay system. Clone #2 was selected for further use.
5
CRISPR/Cas9 Genome Editing: IKKα, IKKβ, c-Myc
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Single guide RNA (sgRNA) sequences for CRISPR/Cas9 were designed using DeskGen (Desktop Genetics,http://www.deskgen.com/ ) using only high-efficiency sgRNAs, avoiding off-target binding in coding regions. The following sgRNA oligonucleotides were used: IKKα: 5′-ACAGACGTTCCCGAAGCCGC-3′ (GeneID: 1147),
IKKβ: 5′-GCTGACCCACCCCAATGTGG-3′ (GeneID: 3551),
c-Myc: 5′-TTTTCGGGTAGTGGAAAACC-3′ (GeneID: 4609).
To generate CRISPR/Cas9 KO plasmids, complementary oligonucleotides for sgRNAs were annealed and ligated into the BbsI-digested (New England Biolabs) pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene plasmid #62988, Addgene) [20 (link)] plasmid. All sequences were verified by sequencing prior to experimental use. Cells were transfected with pSpCas9(BB)-2A-Puro (PX459) V2.0 [20 (link)] bearing respective sgRNA inserts, followed by treatment with 2–4 μg/ml of puromycin 1 day after transfection for further 48 h. After ~ 2 weeks, cell colonies were isolated using glass cloning cylinders (Sigma-Aldrich) and successful genome edits were analyzed by DNA sequencing, Western blot and quantitative PCR (qPCR).
IKKβ: 5′-GCTGACCCACCCCAATGTGG-3′ (GeneID: 3551),
c-Myc: 5′-TTTTCGGGTAGTGGAAAACC-3′ (GeneID: 4609).
To generate CRISPR/Cas9 KO plasmids, complementary oligonucleotides for sgRNAs were annealed and ligated into the BbsI-digested (New England Biolabs) pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene plasmid #62988, Addgene) [20 (link)] plasmid. All sequences were verified by sequencing prior to experimental use. Cells were transfected with pSpCas9(BB)-2A-Puro (PX459) V2.0 [20 (link)] bearing respective sgRNA inserts, followed by treatment with 2–4 μg/ml of puromycin 1 day after transfection for further 48 h. After ~ 2 weeks, cell colonies were isolated using glass cloning cylinders (Sigma-Aldrich) and successful genome edits were analyzed by DNA sequencing, Western blot and quantitative PCR (qPCR).
6
Inducible TALE-Histone Demethylase Cell Lines
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The TALE proteins, produced in-house by the TACGENE platform, are composed of the N-Terminal Region (NTR) and the Central Repeat Domain (CRD) with 17.5 repeated motifs binding the following α-satellite sequence from D7Z1 centromeric region: 5' TGCAATTGTCCTCAAATC 3'. In the N-terminal part of the TALE, the NLS from SV40 and three HA tags were added. At the C-terminal part of the TALE was fused either the cDNA of the wild-type histone lysine demethylase hJMJD2B (kindly given by Sophie Cell lines were generated from U2OS cells using the Tet-On ® system from Clontech ® and following the manufacturer's instructions. In these stable cell lines, two plasmids were integrated successively. The first expresses the transactivator protein under a CMV promoter and possess a G418 resistance gene for selection. The second contains our TALE fusion protein (TALE-hJMJD2B or TALE-GFP) expressed under the PTRE3G promoter containing seven Tet-O repeats and was co-introduced in cells with a linear puromycin marker for selection. Individual colonies were isolated using 150 µL glass cloning cylinders from Sigma-Aldrich ® . The list of plasmids we used is presented in Table S2.
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