Longamp hot start taq dna polymerase

Reverse transcription of the circular miRNA–adapter templates was performed with SuperScript IV (Invitrogen). The reaction mix included 22 μl from the circularization reaction, 1× SSIV Buffer (Invitrogen), 40 units of RNase OUT (Life Technologies), 1.25 μM RT primer, 5 mM dNTPs, and 200 units of SuperScript IV in a 40 μl total reaction volume. The reaction mix was incubated for 30 min at 50 °C followed by 10 min at 80 °C. PCR was performed with LongAmp® Hot Start Taq DNA polymerase (NEB). The reaction included 40 μl from the RT reaction, 1× LongAmp® Taq Reaction Buffer (NEB), 3 mM dNTPs, 0.7 μM forward PCR primer, 0.7 μM reverse index primer, and 10 units of LongAmp® Hot Start Taq DNA polymerase in a 100 μl reaction volume. The PCR reaction was performed for either 5 cycles for the miRXplore pool, or 7, 10, 13, or 16 cycles for 1 μg, 100 ng, 10 ng or 1 ng 3RNA samples respectively. PCR included a first step at 94 °C for 30 s, and 5, 7, 10, 13, or 16 cycles of 94 °C for 15 s, 62 °C for 30 s, and 70 °C for 15 s, with a final step at 70 °C for 5 min.

For S-HUDEP2, edited cells were single-cell sorted into 96-well plates and expanded for 2 weeks. gDNA from each clone was extracted using QuickExtract DNA extraction solution. For SCD HSPCs, edited cells were cultured on semisolid methylcellulose-based medium [MethoCult H4435 Enriched (STEMCELL Technologies, 04445)] for 14 days before being assayed as previously described (7 (link)). Cells from each colony were resuspended in 20 μl of QuickExtract DNA extraction solution for gDNA extraction and processed for S-R NGS for colony genotyping (7 (link)). CRISPR-Cas9 genome editing outcomes were analyzed using CRISPResso2 (30 (link)). To identify clones carrying a LD, the 5.44-kb region containing the on-target cut site at the center was amplified using L-R PCR [LongAmp Hot Start Taq DNA Polymerase; New England Biolabs (NEB), M0534S]. L-R PCR amplicons were run on an agarose gel to check for the gel shift indicating LD. The presence of the LD allele was validated using ddPCR copy number analysis.

Extracted sample DNA was prepared for metagenomic sequencing using the Oxford Nanopore SQK-PBK004 kit (Oxford Nanopore, Oxford, England). After end repair and barcode ligation, sample DNA was amplified with LongAmp Hot Start Taq DNA Polymerase (New England Biolabs, Ipswich, Massachusetts, USA) using the following cycling conditions: 3 min. denaturation at 95°C, 14 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 65°C for 6 min 40 s, and a final extension step at 65C for 6 min. The final fragment size of each library was determined with a Genomic DNA ScreenTape on a 4150 TapeStation System (Agilent, Santa Clara, California, USA) and then pooled in equimolar concentrations in groups of 4 to 5 samples. Sequencing was performed on a GridION Mk1 for 72 h per run using FLO-MIN106 R9.4.1 flow cells.
Library preparation for BRD pathogen isolates was performed with the ONT ligation kit SQK-LSK109 and native barcoding kits EXP-NBD104 and EXP-NBD114 as per manufacture’s instructions. Barcoded isolate DNA was pooled into one library and quantified with the Qubit HS dsDNA Assay kit. 200 ng of prepared library was loaded onto a FLO-MIN106 flow cell and sequenced on an ONT GridION device for 72 h.