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Indexed primers

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Indexed primers are DNA oligonucleotides designed for use in next-generation sequencing (NGS) library preparation. They contain a unique sequence (index) that allows for sample identification and multiplexing during sequencing.

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Lab products found in correlation

Hiseq 2500 platform, by Illumina (2 mentions) Nebnext 2 pcr master mix, by New England Biolabs (2 mentions) Mightyamp dna polymerase, by Takara Bio (2 mentions) Ltp library preparation kit, by New England Biolabs (2 mentions) Nextseq 500, by Illumina (1 mentions) Spriselect magnetic beads, by Beckman Coulter (1 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)

5 protocols using indexed primers

1

TELP Library Preparation for Single-End Sequencing

Cited 1 time
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The fC-CET-enriched genomic DNAs were used directly for library preparation using the TELP protocol23 (link). A minor modification was the use of MightyAmp DNA Polymerase (TAKARA) for one round of on-bead primer extension before PCR amplification. The adaptor-ligated samples were then PCR amplified using NEBNext 2× PCR Master Mix (NEB) and indexed primers (NEB). Libraries were checked using the Agilent 2100 Bioanalyzer before loading onto the Illumina Hiseq 2500 platform. A single-end (100 bp or longer) sequencing mode was suggested for maximal data collection.
Two biological replicates of each mESCs were prepared and sequenced, which means that in parallel, two non-enriched input DNAs (Input: preAI), two AI labeled samples (Input: AI) and two pull-down output samples were sequenced simultaneously following the same procedure.
Xia B., Han D., Lu X., Sun Z., Zhou A., Yin Q., Zeng H., Liu M., Jiang X., Xie W., He C, & Yi C. (2015). Bisulfite-free and Base-resolution Analysis of 5-formylcytosine at Whole-genome Scale. Nature methods, 12(11), 1047-1050.
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2

TELP Library Preparation for Single-End Sequencing

Cited 1 time
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The fC-CET-enriched genomic DNAs were used directly for library preparation using the TELP protocol23 (link). A minor modification was the use of MightyAmp DNA Polymerase (TAKARA) for one round of on-bead primer extension before PCR amplification. The adaptor-ligated samples were then PCR amplified using NEBNext 2× PCR Master Mix (NEB) and indexed primers (NEB). Libraries were checked using the Agilent 2100 Bioanalyzer before loading onto the Illumina Hiseq 2500 platform. A single-end (100 bp or longer) sequencing mode was suggested for maximal data collection.
Two biological replicates of each mESCs were prepared and sequenced, which means that in parallel, two non-enriched input DNAs (Input: preAI), two AI labeled samples (Input: AI) and two pull-down output samples were sequenced simultaneously following the same procedure.
Xia B., Han D., Lu X., Sun Z., Zhou A., Yin Q., Zeng H., Liu M., Jiang X., Xie W., He C, & Yi C. (2015). Bisulfite-free and Base-resolution Analysis of 5-formylcytosine at Whole-genome Scale. Nature methods, 12(11), 1047-1050.
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3

RNA-Seq analysis of HFF cell infection

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HFF cells (1x105) were infected at an MOI of 1 for 72 h. DNA was extracted from infected cells and sheared using a Covaris S220 to an approximate size of 450 bp. Sequencing libraries were created using a Kapa LTP Library preparation kit according to the manufacturer’s instructions, employing indexed primers (New England Biolabs) for PCR. The libraries were sequenced on a NextSeq Mid Output 300 cycle cartridge to produce approximately 5 million paired-end reads of 150 nucleotides (nt). Sequence data have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB58764.
Lista M.J., Witney A.A., Nichols J., Davison A.J., Wilson H., Latham K.A., Ravenhill B.J., Nightingale K., Stanton R.J., Weekes M.P., Neil S.J., Swanson C.M, & Strang B.L. (2023). Strain-Dependent Restriction of Human Cytomegalovirus by Zinc Finger Antiviral Proteins. Journal of Virology, 97(3), e01846-22.
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4

HFF Cell Infection and Sequencing

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HFF cells (1 × 105) were infected at an MOI of 1 for 72 h. DNA was extracted from infected cells and sheared using a Covaris S220 to an approximate size of 450 bp. Sequencing libraries were created using a Kapa LTP Library preparation kit according to the manufacturer’s instructions, employing indexed primers (New England Biolabs) for PCR. The libraries were sequenced on a NextSeq Mid Output 300 cycle cartridge to produce approximately 5 million paired-end reads of 150 nucleotides (nt).
Lista M.J., Witney A.A., Nichols J., Davison A.J., Wilson H., Latham K.A., Ravenhill B.J., Nightingale K., Stanton R.J., Weekes M.P., Neil S.J., Swanson C.M, & Strang B.L. (2023). Strain-Dependent Restriction of Human Cytomegalovirus by Zinc Finger Antiviral Proteins. Journal of Virology, 97(3), e01846-22.
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5

ChIP-Seq Library Preparation Protocol

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Qubit dsDNA HS Assay Kit (Invitrogen, Q23851) was used to measure the quantity of ChIP DNA. Then <5 ng ChIP sample DNA or <20 ng input DNA was used for sequencing libraries using the NEB Next Ultra II DNA Library Prep Kit for Illumina (New England BioLabs, E7645). Two-step purification was reduced to one step by skipping step 3.1 per manufacturer's recommendation to improve the quality of the final output library. The DNA library for each sample was PCR amplified for 12 cycles using indexed primers (New England BioLabs, E7335S or E7500S) to barcode samples. Sample products then underwent a double-sided (.65 x -1 x volume) size selection and purification using SPRIselect magnetic beads (Beckman Coulter, B23318). Concentrations were measured using the Qubit HS dsDNA assay then the size and purification of primer dimmers of DNA libraries was verified by gel electrophoresis. Samples were then combined with less than 20 barcoded samples per sequencing run on an Illumina NextSeq500.
Little D.R., Lynch A.M., Yan Y., Akiyama H., Kimura S., & Chen J. (2020). Lung lineage transcription factor NKX2-1 epigenetically resolves opposing cell fates in vivo.
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