50 μL PCR reactions with 3 μg input gDNA per reaction were run using Phusion® Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific) (95°C for 4 min; 30 times: 98°C for 45s, 60°C for 30 s, 72°C for 1 min; 72°C for 7 min) using primers flanking the gRNA insert containing overhang sequenced compatible with Illumina Nextera XT indexing and 8 random nucleotides to increase the diversity of the sequences (LIB_8xN_NGS_FWD and LIB_8xN_NGS_REV listed in Supplemental Table S3 ). Double size selection was performed using Agencourt AMPure XP beads (Beckman Coulter) to exclude primer dimers and genomic DNA. The amplicons were indexed using Nextera XT Index Kit v2 (Illumina) sequence adapters using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) (95°C for 3 min; 8 times: 95°C for 30s, 55°C for 30 s, 72°C for 30 s; 72°C for 5 min) and subjected to a second round of bead-based size exclusion. The resulting library was quantified with Qubit® using the dsDNA HS Assay Kit (Thermo Fisher Scientific) and the fragment size was determined using a 2100 Bioanalyzer Instrument (Agilent) before running the samples on a NextSeq 500 sequencer (Illumina).
Nextera xt indexing
Manufactured by Illumina
Nextera XT indexing is a library preparation technology developed by Illumina. It enables the generation of sequencing-ready libraries from small amounts of input DNA. The core function of Nextera XT indexing is to attach unique index sequences to DNA fragments, allowing for multiplexed sequencing on Illumina platforms.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 500 sequencer, by Illumina (2 mentions)
Kapa hifi hotstart readymix, by Roche (2 mentions)
Nextera xt index kit v2, by Illumina (2 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
Phusion hot start 2 high fidelity dna polymerase, by Thermo Fisher Scientific (2 mentions)
Dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
2100 bioanalyzer instrument, by Agilent Technologies (2 mentions)
2 protocols using nextera xt indexing
1
PCR-based Multiplexed Amplicon Sequencing
2
gRNA Library Preparation for NGS
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50 µL PCR reactions with 3 µg input gDNA per reaction were run using Phusion® Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific) (95°C for 4 min; 30 times: 98°C for 45s, 60°C for 30 s, 72°C for 1 min; 72°C for 7 min) using primers flanking the gRNA insert containing overhang sequenced compatible with Illumina Nextera XT indexing and 8 random nucleotides to increase the diversity of the sequences (LIB_8xN_NGS_FWD and LIB_8xN_NGS_REV listed in Supplemental Table S2). Double size selection was performed using Agencourt AMPure XP beads (Beckman Coulter) to exclude primer dimers and genomic DNA. The amplicons were indexed using Nextera XT Index Kit v2 (Illumina) sequence adapters using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) (95°C for 3 min; 8 times: 95°C for 30s, 55°C for 30 s, 72°C for 30 s; 72°C for 5 min) and subjected to a second round of bead-based size exclusion. The resulting library was quantified with Qubit® using the dsDNA HS Assay Kit (Thermo Fisher Scientific) and the fragment size was determined using a 2100 Bioanalyzer Instrument (Agilent) before running the samples on a NextSeq 500 sequencer (Illumina).
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