An RNeasy kit (Qiagen) was used to purify RNA for RNAseq perturbation and off-target analysis. Samples were normalized to an input of 150 ng before preparation with a NEBNext® Ultra™ II RNA Library Prep (Magnetic Bead) kit (New England Biolabs). The polyA mRNA protocol was used for all preps. Samples were barcoded using Illumina i5 and i7 adaptors from NEBNext® Multiplex Oligos for Illumina (Dual Index Primers Set 1) kit (New England Biolabs) and sequenced using a 150-cycle NextSeq 500/550 High Output Kit v2.5 with 75 forward and 75 reverse reads. The output samples were processed using Galaxy (version 23.1.rc1. FASTQ trimming was performed by Cutadapt, using an R1 minimum length cutoff of 20 and quality cutoff of 20. Trimmed reads were mapped to the human genome release 19 (GRCh37.p13) using STAR with GeneCounts. featureCounts was then used to generate reads per gene, specifying the input as unstranded, using the same GFF file for hg19 used by STAR, GFF feature type filter as “exon”, GFF gene identifier as “gene_id”, and input as “Paired end, count as single fragment”. Read filtering minimum mapping quality per read was set to 10. Differentially expressed genes were calculated using DeSeq2 on the count data. Volcano plots were generated using Galaxy and colored based on a 0.01 FDR.
Nebnext multiplex oligos for dual index primers set 1 kit
Manufactured by Illumina
The NEBNext® Multiplex Oligos for Illumina (Dual Index Primers Set 1) kit contains a set of 24 unique dual index primer pairs that can be used with Illumina sequencing platforms to introduce index sequences during library preparation.
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Lab products found in correlation
2 protocols using nebnext multiplex oligos for dual index primers set 1 kit
1
RNA-seq Library Preparation and Analysis
2
Amplifying gRNA Spacers for Sequencing
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For both Donor and Host Library plasmid pools, PCR using ‘phased primers’ amplified the gRNA spacers to be sequenced. Briefly, five forward and five reverse primers were made, each that bound to the same site of the library plasmids but possessing zero to four additional random bases at the 5′ end; the purpose of these extra bases is to alleviate issues inherent to sequencing amplicons on Illumina platforms, as the very low complexity of amplicon molecules prevents high-quality base calls from being made by the Illumina system. These forward and reverse primer mixes were used together with 30 ng of library vector in a Q5 Hot Start PCR as per manufacturer's instructions. The PCR product was purified on 3% gel and isolated using the NEB Monarch DNA Gel Extraction Kit, quantified using the Promega Quantus fluorometer and the QuantiFluor ONE dsDNA System, and then used in the NEBNext Ultra II DNA Library Prep Kit for Illumina. During the amplification step, one forward and one reverse primer per library from the NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) kit was used to complete the protocol.
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