Directpcr lysis reagent

The generation of mice with tamoxifen-inducible, Tie2.Cre-ER^T2-mediated deletion of LepR in endothelial cells was described previously^{12 (link),17 (link)}. For Cre recombinase activation, mice (6 weeks-of-age) were fed tamoxifen citrate-containing rodent chow (Envigo; TD.130860) for 6 weeks^{49 (link)}. Genomic DNA from the brain, lung, small intestine, subcutaneous adipose tissue (SCAT) and visceral adipose tissue (VAT) was isolated using Direct PCR lysis reagent (Peqlab) containing 0.2 mg/mL proteinase K (Peqlab). The mouse genotype was determined using the following primers: Tie2.Cre (Tie2Cre1: 5′-CGA GTG ATG AGG TTC GCA AG-3′; Tie2Cre2: 5′-TGA GTG AAC GAA CCT GGT CG-3′); LepR (LepRflox1: 5′-GTC ACC TAG GTT AAT GTA TTC-3′; LepRflox2: 5′-TCT AGC CCT CCA GCA CTG GAC-3′. Tamoxifen-induced deletion of LepR gene was confirmed using the primers: LepRflox1: 5′-GTC ACC TAG GTT AAT GTA TTC-3′ and LepR delta/∆: 5′-GCA ATT CAT ATC AAA ACG CC-3′). Sex-matched littermate Tie2.ER^T2-WT LepR^flox/flox mice, fed tamoxifen-containing rodent diet, were used as controls throughout the entire study.

Genotyping was accomplished by isolating genomic DNA from tail tips, using DirectPCR Lysis Reagent (Peqlab) and following PCR and DNA digestion by restriction enzyme PpuMI (New England Biolabs).
The following primers were used:

5′-TCTGGGAGTAAGGCCAAAGAC-3′ (forward) and

5′-TGAGCTCAAAGACAGCAGACC-3′ (reverse).

PCR was performed for 35 cycles, 95°C for denaturation, 60°C for annealing, and 72°C for elongation performed with DNA Taq Polymerase (New England Biolabs) and a PeqStar Thermocycler (Peqlab). An aliquot of the PCR reaction was used to validate the presence or absence of the constitutive knock-in. DNA was digested using PpuMI. 10 U per enzyme were pipetted into the PCR reaction aliquot and incubated at the appropriate temperature for 48 h. A control plasmid demonstrated that the digest worked successfully.
The 543-bp PCR product was unaffected in wild types, partially digested in heterozygous mice, and completely digested in ADAM17^3x/3x mice into two fragments (363 and 180 bp) as detected on an agarose gel.

After removing the medium, the wells were washed twice with PBS. Subsequently, 70 μL of the Direct PCR^® lysis reagent (PeqLab, #Viag401‐E) supplemented with 0.2 mg/mL fresh proteinase K together with 70μl water was added to each well containing adhesive living cells. The solution in the wells was pipetted up and down several times using a multichannel pipet. The 96‐well plate was then placed on a heater at 60°C overnight to lyse the cells. On the next day, several wells were checked under a microscope to ensure complete lysis. The plates were then heated at 85°C for 45 minutes on the heater to inactivate the proteinase K. Afterward, the plates were loaded into a centrifuge to spin down the debris at 250 g for 1‐2 minute. Supernatants containing the extracted DNA were further purified by precipitation.

Ear biopsies were digested for 2–16 h at 55 °C in 50 µl of the DirectPCR Lysis Reagent (Peqlab, 31-102-T) plus 2 µl of Proteinase K (Roche), followed by 45 min at 85 °C. Supernatants of BAFF-E247K ki lysates were used for PCR using primers fwd 5ʹ-ACCCTGTTCCGATGTATTCA-3ʹ and rev 5ʹ-TAAGAGAGTGCCAGGTCCC-3ʹ, with 30 cycles of 94 °C (7 s)/58 °C (20 s)/72 °C (40 s). Supernatants of BAFF-ko lysates were used for PCR using primers wt shared 5ʹ-GCAGATTGAGCAATCCATGGAAGGCCA-3ʹ, wt specific 5ʹ-CAAGTTGATGTCCTGACCCAAGGCACC-3ʹ, and neo 5ʹ-TGGCAGGGTCTTTGCAGACTCATCCAT-3ʹ, with 30 cycles of 94 °C (7 s)/60 °C (20 s)/72 °C (20 s). For the screening of recombined ES cells, genomic DNA was amplified with primers NeoR fwd 5ʹ-CCTTCTATCGCCTTCTTGAC-3ʹ and BAFF rev 5ʹ-GTGGAACAGATAAGGTGCCT-3ʹ, with 3 cycles of 95 °C (30 s)/64 °C [ramp-0.5 °C/cycle] (30 s)/68 °C (2 min), followed by 30 cycles of 95 °C (30 s)/54 °C (30 s)/68 °C (2 min), using TaKaRa LA polymerase (TaKaRa) at 5 U/ml.

Cells were lysed in DirectPCR lysis reagent (PeqLab) supplemented with 200 μg/ml proteinase K (Roche Diagnostics) and the edited loci were amplified by PCR. The PCR primers carried multiplexing barcodes and the partial Illumina adapters as overhangs. The resulting amplicons were analyzed by gel electrophoresis and subsequently DNA was isolated with a QIAquick Gel Extraction Kit (Qiagen). The DNA concentration was adjusted to 20 ng/μl and up to six barcoded samples were pooled. Amplicon sequencing was performed by the Genewiz Amplicon-EZ service. The data was analysed using the Sabre (https://github.com/najoshi/sabre) and CRISPresso 2.0 (https://github.com/pinellolab/CRISPResso2 (57 (link))) packages in order to determine the indel frequencies.