DNA was extracted from FACS-sorted cells using the Qiagen DNeasy Blood & Tissue Kit and 500 ng of genomic DNA was spiked with 0.5% (w/w) of unmethylated lambda phage DNA (Promega) for calculation of the bisulfite non-conversion rate and bisulfite converted with the EZ DNA Methylation-Direct Kit (Zymo Research). PCR amplicons were designed with methprimer and bisulfite-converted DNA was amplified with 40 cycles of PCR using MyTaq HS mix (Bioline). PCR reactions were pooled and purified with SPRI beads. One microgram of pooled PCR products in 14.5 μl were phosphorylated by adding 15 μl 2X Quick Ligase Buffer (New England Biolabs) and 0.5 μl T4 polynucleotide kinase (New England Biolabs) and incubating at 37°C for 30 min. Illumina TruSeq adapters synthesized by IDT and annealed by heating to 99°C and slowly cooling to 20°C were ligated to the phosphorylated PCR products by adding 3.75 μl 10 μM annealed TruSeq adapters, 10 μl 2X Quick Ligase Buffer (New England Biolabs), and 6 μl water. The ligation reactions were incubated at 25°C for 20 min and stopped by adding 2 μl 0.5 M EDTA. The DNA was purified by adding 20.8 μl (0.4 volumes) of SPRI beads. The libraries were subjected to single-end 300 cycle sequencing on the Illumina MiSeq.
Quick ligase buffer
Manufactured by New England Biolabs
Sourced in United States
The Quick Ligase Buffer is a reagent used in molecular biology experiments. It is designed to facilitate the ligation of DNA fragments, a process where DNA molecules are joined together. The buffer provides the optimal ionic conditions and cofactors required for the efficient activity of DNA ligase enzymes, which catalyze the formation of phosphodiester bonds between the ends of DNA strands.
Automatically generated - may contain errors
Lab products found in correlation
T4 ligation buffer, by New England Biolabs (4 mentions)
T4 polynucleotide kinase, by New England Biolabs (3 mentions)
Quick ligase, by New England Biolabs (3 mentions)
T4 pnk, by New England Biolabs (2 mentions)
Stable competent e coli, by New England Biolabs (2 mentions)
Ez dna methylation direct kit, by Zymo Research (1 mentions)
Mytaq hs mix, by Meridian Bioscience (1 mentions)
Unmethylated lambda phage dna, by Promega (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Lenticrispr v2 plasmid, by Addgene (1 mentions)
Lenticrispr v2, by New England Biolabs (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Ampure bead, by Beckman Coulter (1 mentions)
Epitect bisulfite kit, by Qiagen (1 mentions)
T4 dna ligase buffer, by New England Biolabs (1 mentions)
Ex taq dna polymerase, by Takara Bio (1 mentions)
Klenow dna polymerase, by New England Biolabs (1 mentions)
Klenow buffer, by New England Biolabs (1 mentions)
Klenow exo minus, by New England Biolabs (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
11 protocols using quick ligase buffer
1
Bisulfite Sequencing of FACS-sorted Cells
2
Lentiviral CRISPR Plasmid Cloning
3
Bisulfite Sequencing of Moss Protonema DNA
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Around 0.5 μg of genomic DNA from protonema tissue was extracted,sheared (by sonication), end repaired (10 µl T4 DNA ligase buffer (NEB B0202S), 4 µl 10 mM dNTP mix, 1 µl T4 DNA polymerase (NEB M0203S), 1 µl Klenow DNA polymerase (NEB M0210S), 1 µl T4 PNK (NEB M0201S), water to 100 µl), A-tailed (5 µl Klenow buffer (NEB2), 10 µl 1 mM dATP, 1 µl Klenow exo minus (NEB M0212S), water to 50 µl), and ligated to methylated-adapters (25 µl quick ligase buffer (NEB), 1 µl 10 mM preannealed bs-seq-adapters (Supplementary Table 2 ), 1 µl DNA quick ligase (NEB M2200S), water to 50 µl). Adaptor-ligated libraries were subjected to two sequential treatments of bisulfite conversion using the EpiTect Bisulfite kit (Qiagen). Bisulfite-converted libraries were amplified by PCR (2.5 U of ExTaq DNA polymerase (Takara Bio), 5 µl of 10x Extaq reaction buffer, 25 mM dNTPs, 1 µl bs-seq-primers (Supplementary Table 2 ), and adding water to 50 µl). PCR program: 95 °C for 3 min, then 12–14 cycles of 95 °C for 30 s, 65 °C for 30 s, and 72 °C for 60 s. Between library preparation steps and following PCR, DNA was purified with the solid-phase reversible immobilization method using AM-Pure beads (Beckman Coulter) and quantified with Bioanalyzer (Agilent). Deep sequencing was performed on Illumina Hi-Seq 2000.
4
CRISPR-Cas9 Knockdown of TAZ/YAP and Aldh1a1 Genes
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For TAZ/YAP or Aldh1a1 gene knockout, 1–2 guided RNAs (gRNAs) sequences targeting TAZ/YAP or Aldh1a1 were chosen (Supplementary Table 2 ) based on published whole-genome gRNA libraray sequences [35 (link), 36 (link)] and single-stranded complementary oligos with BsmBI overhang were synthesized (McGill University). FastDigest BsmBI and FastAP from Fermentas were used to digest LentiCRISPR v1 (Addgene) lentiviral vector. Digested vectors were purified by QIAquick Gel Extraction Kit and eluted in EB buffer. Oligos were phosphorylated and annealed by T4 polynucleotide kinase (PNK, NEB, #M0201S) in T4 ligation Buffer (NEB) following a thermocycler running at 37°C for 30 minutes, 90°C for 5 minutes and then ramp down to 25°C at 5°C per minute. Ligation was catalyzed by mixing annealed oligos and digested LentiCRIPSR v1 vector with Quick Ligase in Quick Ligase Buffer (NEB, #M2200S), followed by transformation into Stbl3 bacteria. Production of lentivirus and establishment of stable cell lines were as described above.
5
CRISPR gRNA Cassette Cloning
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gRNA cassettes were annealed from two oligonucleotides (top: 5’-CACCG(N)20-3’, bottom: 5’-AAAC(N)20C-3’) by combining 1 μl of each 100 μM oligonucleotide with 1 μl of 10x T4 ligation buffer (NEB cat. no. B0202S), 6.5 μl of water, and 0.5 μl of T4 polynucleotide kinase (NEB cat. no. M0201S), incubating as follows: 37 °C for 30 min (oligonucleotide phosphorylation), 95 °C for 5 min, then ramping from 90 °C to 25 °C at 5 °C/min. Plasmid backbone was prepared by digesting 1 μg of CROPseq-Guide-Puro with 10 units of BsmBI (NEB cat. no. R0580L) in a volume of 30 μl 1x NEB buffer 3.1, incubating for 1 hour at 55 °C. To dephosphorylate the digested plasmid, we added 2 μl of shrimp alkaline phosphatase (rSAP, NEB cat. no. M0371L), incubating for 1 hour at 37 °C followed by heat inactivation (both BsmBI and rSAP) for 20 min at 80 °C. Ligation reactions were set up as follows: 1.6 μl of rSAP reaction, 1 μl gRNA cassette (diluted 1:200 in water), 5 μl 2x Quick Ligase buffer, 2.4 μl water, and 1 μl Quick Ligase (NEB cat. no. M2200S), incubated at 25 °C for 15 min. The ligation reaction was chemically transformed into NEB Stable competent E. coli (NEB cat. no. C3040H) following the manufacturer’s high-efficiency protocol.
6
CRISPR gRNA Cassette Cloning
7
Preparing 16S rRNA Sequencing Libraries
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Sequencing libraries of purified 16S rRNA for E. coli str. MRE600, V. cholerae str. A1552, and M. maripaludis str. S2 were prepared as follows: 2 pmol 16S rRNA adapter and 1.5 μg purified 16S rRNA (approximately 3 pmol) were added to a 15 μL reaction in 1x Quick Ligase buffer with 3000U T4 DNA ligase (New England Biolabs). The reaction was incubated at room temperature for 10 minutes. These reactions were cleaned up using 1.8x volume of RNAclean XP beads (Beckman Coulter), washed once with 80% ethanol and resuspended in 20 μl nuclease-free water. The RNA sequencing adapter (Oxford Nanopore Technologies) was ligated to the RNA library following manufacturer recommended protocol.
8
High-Throughput Barcode Encapsulation
9
High-Throughput Barcode Encapsulation
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Barcodes were commercially synthesized (IDT, USA) and suspended in quick ligase buffer (NEB, USA) at a concentration of 500 uM in 384 well-plates. We use a 96 parallel drop-maker microfluidic chip with aqueous inlets for each drop-maker that precisely fit one quarter of a 384 well-plate and that are immersed in 96 different wells, each containing a unique barcode. Oil with surfactant is distributed to all drop-makers via a common inlet that is connected to a pressurized (9 psi) oil reservoir. The plate and the microfluidic parallel device are placed in a pressure chamber while a common outlet for all 96 barcode drop-makers is located outside the pressure chamber. Upon pressurizing the chamber (6 psi), each of the 96 barcode solutions is forced through its own drop-maker, thereby forming an emulsion of ∼35um diameter drops where every drop contains about 1 billion copies of one of the 96 barcodes. The process is repeated until all barcodes are encapsulated. Before use, the emulsion is pooled in a single tube and mechanically mixed by rolling the tube for 5 minutes.
10
Rapid CRISPR Plasmid Construction Protocol
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BsmBI enzyme was used to digest SadCas9-ABI or SpdCas9-PYL1 plasmids (CLOuD9, System Biosciences, Cat# CASCL9-100A) at 37°C for 12 h. On the next day, the mixture was incubated at 65°C for 20 min to inactivate the enzyme. Run the mixture on 1.5% agarose DNA gel, cut the correct fragments and purify the DNA by Omega DNA purification Kit. The size of SpdCas9-PYL1 and SadCas9-ABI vectors are 14049 bp and 12984 bp respectively (Morgan et al., 2017 (link)).
| Material | Volume |
|---|---|
| dcas9-ABI/PYL vector | 5ug |
| BsmBI (10000 units/mL) | 3ul |
| 10 | 5ul |
| DTT (20 mM) | 1ul |
| H2O | Add to 50ul |
Annealing and phosphorylation of sgRNA oligoes
| Material | Volume |
|---|---|
| Oligo 1 (100uM) | 1 ul |
| Oligo 2 (100uM) | 1 ul |
| 10 | 1 ul |
| T4 PNK (10000 units/mL) | 0.5 ul |
| H2O | 6.5 ul |
| Steps | Temperature | Time | Cycles |
|---|---|---|---|
| 37°C | 30 min | 1 | |
| 95°C | 5 min | 1 | |
| 5 °C/min decrease to 25°C | 14 min | 1 | |
| 12°C | Forever | 1 |
Ligation at 20°C for 10 min, and at 37°C for 10 min
| Material | Volume |
|---|---|
| Digested vectors | 50 ng |
| Annealed Oligoes | 1 ul |
| 2 | 5 ul |
| Quick ligase (NEB M2200) | 1 ul |
| H2O | Add to 11 ul |
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