Cells were grown at 30 °C to an OD600 of 1.0, and DSBs were induced by the addition of 2 % galactose. Cells were harvested after various induction times, and genomic DNA was extracted by vortexing cells with glass beads and phenol. Twenty microliters of genomic DNA (60 ng in 1X Exonuclease I buffer (New England Biolabs)) was digested with 20 units of E. coli exonuclease I at 37 °C overnight. The level of DNA resection adjacent to the specific DSB was measured by qPCR using primers annealing 1.6 kb upstream of the DSB. All values were normalized to values for an independent locus on chromosome 5 (POL5). The assay was repeated three times and reported as mean (±SD) [47 (link)].
Exonuclease 1 buffer
Manufactured by New England Biolabs
Exonuclease I buffer is a solution formulated to maintain the activity and stability of Exonuclease I enzyme. Exonuclease I is an enzyme that selectively digests single-stranded DNA in the 3' to 5' direction.
Automatically generated - may contain errors
Lab products found in correlation
Rnaseout, by Thermo Fisher Scientific (2 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by New England Biolabs (2 mentions)
Dntp mix, by Thermo Fisher Scientific (2 mentions)
Proteinase k, by Qiagen (2 mentions)
Actinomycin d, by Merck Group (2 mentions)
Exonuclease 1, by New England Biolabs (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ssc buffer, by Merck Group (1 mentions)
Pvp 40, by Merck Group (1 mentions)
5 protocols using exonuclease 1 buffer
1
DNA Resection Measurement at DSBs
2
Single-cell RNA-seq with permeabilization
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We followed a protocol as described in Stahl et al12 (link)and Salmén et al21 (link). Briefly, tissue sections were permeabilized using exonuclease I buffer (NEB) for 30 min at 37°C and 0.1X pepsin (pH 1) for 10 min at 37°C, followed by in situ cDNA synthesis overnight at 42°C using Superscript III supplemented with RnaseOUT in 1XFS buffer, 5 mM DTT, 0.5 mM dNTP mix (all from ThermoFisher Scientific), 50 μg/ml actinomycin D in 1% DMSO (Sigma-Aldrich) with 0.19 mg/ml BSA (NEB). For breast cancer samples, the tissue permeabilization steps included a 20 min tissue incubation with 14 U collagenase I in Hank’s balanced salt solution (ThermoFisher Scientific) followed by digestion with 0.1X pepsin (pH 1) for 10 min at 37°C. Tissue sections were digested after cDNA synthesis using proteinase K (Qiagen) for 1h at 56°C and the barcode-transcript information cleaved using a USER (NEB) (for 2h at 37°C). The material was processed into libraries as described in Jemt et al22 (link) and 1.08 pM sequenced on an Illumina Nextseq 500 instrument with v2 chemistry using paired-end 300 bp reads (R1 125 bp and R2 175 bp for MOB; R1 150 bp with R2 150 bp for the breast cancer samples).
3
Optimizing VH Forward Primer Activation
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The VH forward Generation 1 rh-PCR primer pool was diluted to a concentration of 0.8 μM in 125 μL of either 1X Phusion Hi-Fi Buffer (NEB) or 1X Exonuclease I Buffer (NEB). Aliquots of these reactions, 10 μL, were dispensed into replicate wells of three 96-well PCR plates and additions of either 1 μL (2U/μL) Phusion High Fidelity DNA Polymerase (NEB), 1 μL (20U/μL) Exonuclease I (NEB), or 1 μL water were made at T0. The plates were incubated at 37°C and at timepoints of 1 hour, 3 hours, and 6 hours one plate was removed from incubation and 2 μL of the reactions were analyzed by capillary electrophoresis as described above.
4
Single-cell RNA-seq with permeabilization
5
Tissue Permeabilization for Spatial Transcriptomics
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To pre-permeabilize the tissue, the slides were mounted in a plastic cassette and sections incubated in pre-permeabilization solution (24 (link)) (48 μl Exonuclease I buffer, NEB, cat no. B0293S; 4.5 μl of BSA, Sigma-Aldrich, cat no. A7039-100G; and 2% (w/v) PVP40, Sigma-Aldrich, cat no. PVP40-1KG) at 37°C for 30 min. This was followed with a wash with 0.1 × SSC buffer (Sigma-Aldrich, cat no. S6639L). The sections were permeabilized with Permeabilization mix™ (10x Genomics) at 37°C for different times (1, 3, 6, 15, 30 min TO slides) or for 3 min (GE slides). Then, wells were washed with 0.1× SSC buffer. After permeabilization, Reverse transcription mixture™ (10x Genomics) was added to each section and incubated at 56°C for 45 min as described in the 10x Genomics User guide (PN-1000186, CG000239_VisiumSpatialGeneExpression_UserGuide_RevD).
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