5 adapter, by Illumina - Product details

1

Yeast cell RNA extraction and 5'RACE

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Yeast cells were grown to stationary phase and regrown to mid-exponential phase in SCD-URA medium (as was done prior to expression measurements by FACS; see above). Cells were aliquotted to 25 mL and centrifuged to pellet cells at 3000g for 8 min. The growth medium was removed, and total RNA was extracted using lyticase digestion followed by the TriReagent (MRC) RNA extraction protocol.
RNA libraries for transcription start site mapping (5′ end RNA-seq) were prepared as in Wurtzel et al. (2010) (link). In brief, RNA was incubated with tobacco acid pyrophosphatase (TAP, Epicentre) to treat 5′ ends, and 3′ ends were blocked using NaOI4. Illumina's 5′ adapter was ligated to the RNA with T4 RNA ligase (NEB). cDNA priming was done using a YFP gene-specific primer (GSP). Following cDNA synthesis, YFP amplicons were amplified for 18 cycles using a nested YFP GSP attached to an Illumina 3′ adapter and a 5′ Illumina adapter as forward primer.

Lubliner S., Regev I., Lotan-Pompan M., Edelheit S., Weinberger A, & Segal E. (2015). Core promoter sequence in yeast is a major determinant of expression level. Genome Research, 25(7), 1008-1017.

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2

Vaginal Microbiome DNA Extraction and Amplification Protocol

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The total DNA from vaginal swabs was extracted using the modified protocol MoBio Powersoil modified Method #3 as previously published (Mattei et al., 2019 (link)). DNA concentration was measured using Nanodrop.
The V1-V3 regions of the 16S rDNA were amplified using forward primers: 27F with 12 bp golay barcodes containing a specific Illumina 5’ adapter for each sample and a common reverse primer 515 R (Mattei et al., 2019 (link)). In brief, PCR was performed in triplicate in a 50 μl reaction mixture containing 10 ng of template DNA and 2x Phusion HotStart Ready Mix. The following thermal cycling conditions were used: 5 min of initial denaturation at 94°C; 25 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 30 s, and elongation at 72°C for 30 s; and the last step at 72°C for 10 min. The amplified PCR products of approximately 650 bp in size from each sample were pooled in equimolar concentrations. This pooled PCR product was purified using AgenCourt AMPure XP magnetic beads. High throughput sequencing was performed on an Illumina MiSeq 2 × 300 platform (Illumina, Inc. San Diego) in accordance with the manufacturer’s instructions. Image analysis and base calling were carried out directly on the MiSeq.

Kumar M., Murugesan S., Singh P., Saadaoui M., Elhag D.A., Terranegra A., Kabeer B.S., Marr A.K., Kino T., Brummaier T., McGready R., Nosten F., Chaussabel D, & Al Khodor S. (2021). Vaginal Microbiota and Cytokine Levels Predict Preterm Delivery in Asian Women. Frontiers in Cellular and Infection Microbiology, 11, 639665.

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3

Vaginal Microbiome 16S rDNA Profiling

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The total DNA from vaginal swabs was extracted using the modified protocol MoBio Powersoil modified Method #3 as previously published [27] . DNA concentration was measured using Nanodrop.
The V1-V3 regions of the 16S rDNA were amplified using forward primers: 27F with 12 bp golay barcodes containing a specific Illumina 5' adapter for each sample and a common reverse primer 515 R [27] . In brief, PCR was performed in triplicate in a 50 μL reaction mixture containing 10 ng of template DNA and 2x Phusion HotStart Ready Mix. The following thermal cycling conditions were used: 5 min of initial denaturation at 94 °C; 25 cycles of denaturation at 94 °C for 30 s, annealing at 62 °C for 30 s, and elongation at 72 °C for 30 s; and the last step at 72 °C for 10 min. The amplified PCR products of approximately 650 bp in size from each sample were pooled in equimolar concentrations. This pooled PCR product was purified using AgenCourt AMPure XP magnetic beads. High throughput sequencing was performed on an Illumina MiSeq 2 × 300 platform (Illumina, Inc. San Diego) in accordance with the manufacturer's instructions. Image analysis and base calling were carried out directly on the MiSeq.

Kumar M., Murugesan S., Singh P., Saadaoui M., Elhag D.A., Terranegra A., Kabeer B.S.A., Marr A.K., Kino T., Brummaier T., McGready R., White N.J., Chaussabel D., & Khodor S.A. (2020). Vaginal microbiota and cytokine levels predict preterm delivery in Asian women.

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4

Single-cell RNA-seq using CEL-Seq2

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Single S-EGFP or W-EGFP cells were plated by FACS (SH800, Sony, Tokyo, Japan) into individual wells of a 384-plate (Piko PCR Plate, Thermo Fisher Scientific) pre-loaded with lysis buffer. The CEL-Seq2 protocol established by Hashimshony et al.^{37 (link)} was used for RNA extraction and library preparation. Briefly, the RNA of each cell was reverse transcribed using CEL-Seq primers containing an anchored poly(T), a 6-bp unique molecular identifier, a 5’ Illumina adapter (San Diego, CA, USA), a cell-specific 6-bp barcode, and a T7 promoter (Supplementary Table 2). The External RNA Controls Consortium spike-ins (Thermo Fisher Scientific) were added to each preparation. After second-strand synthesis reaction, the double-stranded cDNAs were transcribed in vitro by T7 RNA polymerase. The synthesized RNAs were reverse transcribed using random primers with the 3’ Illumina adapter. Finally, the libraries were amplified by PCR (11 cycles). The pair-ended CEL-Seq2 libraries were sequenced by HiSeq 2500 (Illumina).

Inoue M., Baba T., Takahashi F., Terao M., Yanai S., Shima Y., Saito D., Sugihara K., Miura T., Takada S., Suyama M., Ohkawa Y, & Morohashi K.I. (2022). Tmsb10 triggers fetal Leydig differentiation by suppressing the RAS/ERK pathway. Communications Biology, 5, 974.

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5

Adapter Design for Single-Cell Sequencing

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The adapter was designed (5’ to 3’) with a 4 nt fork, a T7 promoter, the 5’ Illumina adapter (as used in the Illumina small RNA kit), a 3 nt UMI (unique molecular identifier), an 8 nt unique barcode followed by CA. The Dam-RING1B mESCs were processed with different adapters. These contained a 6 nt fork, a 6 nt unique barcode followed by GA. The barcodes were designed with a hamming distance of at least two between them. Bottom sequences contained a phosphorylation site at the 5’ end. Adapters were produced as standard desalted oligos. Top and bottom sequences were annealed at a 1:1 volume ratio in annealing buffer (10 mM Tris pH 7.5–8.0, 50 mM NaCl, 1 mM EDTA) by immersing tubes in boiling water, then allowing to cool to room temperature. The oligo sequences can be found in Supplementary Table 2.

Rooijers K., Markodimitraki C.M., Rang F.J., de Vries S.S., Chialastri A., de Luca K.L., Mooijman D., Dey S.S, & Kind J. (2019). scDam&T‐seq combines DNA adenine methyltransferase-based labeling of protein-DNA contact sites with transcriptome sequencing to analyze regulatory programs in single cells. Nature biotechnology, 37(7), 766-772.

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6

Single-cell RNA-seq library preparation

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After sorting, single cell TRIzol extractions were performed and the dried RNA pellet was resuspended in primer solution, containing the 5’ Illumina adapter, a cell specific barcode, a unique molecular identifier (UMI), the T7 promoter and a poly T stretch. The RNA-primer solution was briefly denatured, cooled on ice and subsequently first strand synthesis mix was added. After the first strand synthesis reaction the DNA:RNA hybrids were converted into dsDNA, cleaned up and subsequently o/n in vitro transcription was performed. The obtained RNA was thereafter fragmented and Illumina compatible libraries were made using the TruSeq small RNA sample prep kit. (Grün et al., 2014 (link)).

Roovers E.F., Kaaij L.J., Redl S., Bronkhorst A.W., Wiebrands K., de Jesus Domingues A.M., Huang H.Y., Han C.T., Riemer S., Dosch R., Salvenmoser W., Grün D., Butter F., van Oudenaarden A, & Ketting R.F. (2018). Tdrd6a Regulates the Aggregation of Buc into Functional Subcellular Compartments that Drive Germ Cell Specification. Developmental Cell, 46(3), 285-301.e9.

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7

RT Primer Design for Illumina Sequencing

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The RT primer was designed according to the Yanai protocol23 (link) with an anchored polyT, an 8 nt unique barcode, a 6 nt UMI (unique molecular identifier), the 5’ Illumina adapter (as used in the Illumina small RNA kit) and a T7 promoter. The barcodes were designed with a hamming distance of at least two between them. Primers are desalted at the lowest possible scale, stock solution 1 μg/μL. The oligo sequences can be found in Supplementary Table 3.

Rooijers K., Markodimitraki C.M., Rang F.J., de Vries S.S., Chialastri A., de Luca K.L., Mooijman D., Dey S.S, & Kind J. (2019). scDam&T‐seq combines DNA adenine methyltransferase-based labeling of protein-DNA contact sites with transcriptome sequencing to analyze regulatory programs in single cells. Nature biotechnology, 37(7), 766-772.

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8

Adapter Design for Single-Cell Sequencing

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The adapter was designed (5’ to 3’) with a 4 nt fork, a T7 promoter, the 5’ Illumina adapter (as used in the Illumina small RNA kit), a 3 nt UMI (unique molecular identifier), an 8 nt unique barcode followed by CA. The Dam-RING1B mESCs were processed with different adapters. These contained a 6 nt fork, a 6 nt unique barcode followed by GA. The barcodes were designed with a hamming distance of at least two between them. Bottom sequences contained a phosphorylation site at the 5’ end. Adapters were produced as standard desalted oligos. Top and bottom sequences were annealed at a 1:1 volume ratio in annealing buffer (10 mM Tris pH 7.5–8.0, 50 mM NaCl, 1 mM EDTA) by immersing tubes in boiling water, then allowing to cool to room temperature. The oligo sequences can be found in Supplementary Table 2.

Rooijers K., Markodimitraki C.M., Rang F.J., de Vries S.S., Chialastri A., de Luca K.L., Mooijman D., Dey S.S, & Kind J. (2019). scDam&T‐seq combines DNA adenine methyltransferase-based labeling of protein-DNA contact sites with transcriptome sequencing to analyze regulatory programs in single cells. Nature biotechnology, 37(7), 766-772.

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9

Microbiome Analysis of Fecal Samples

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Fecal microbial DNA was extracted from each sample with the QIAamp PowerFecal Pro Kit (Qiagen, Germany) following the manufacturer’s instructions. The quantity of the extracted DNA was measured using Qubit Flex Fluorometer (Thermo Fisher Scientific, USA). The 16S rRNA gene of the samples was amplified at the V4-V5 hypervariable region by using the 515F (5′-barcoded-CGCTCTTCCGATCTGTGNCAGCMGCCGCGGTRA-3′) forward primer attached to a 5′ Illumina adapter and the 907R (5′-barcode-GTGCTCTTCCGATCCGYCWATTYHTTTRAGTTT-3′) indexed reverse primer. Amplicon sizes and concentrations were measured using an Agilent 2100 bioanalyzer (Agilent Technologies, USA) and Qubit Flex Fluorometer (Thermo Fisher Scientific, USA), respectively. Each amplified 16S rRNA gene library was diluted and pooled together. Sequencing was performed using the Illumina Miseq platform with MiSeq Reagent Kit v2 (300-cycle kits) at the KNU NGS Core Facility (Daegu, South Korea).

Kim M.J., Jung D.R., Lee J.M., Kim I., Son H., Kim E.S, & Shin J.H. (2023). Microbial dysbiosis index for assessing colitis status in mouse models: A systematic review and meta-analysis. iScience, 27(1), 108657.

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10

RT Primer Design for Illumina Sequencing

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The RT primer was designed according to the Yanai protocol23 (link) with an anchored polyT, an 8 nt unique barcode, a 6 nt UMI (unique molecular identifier), the 5’ Illumina adapter (as used in the Illumina small RNA kit) and a T7 promoter. The barcodes were designed with a hamming distance of at least two between them. Primers are desalted at the lowest possible scale, stock solution 1 μg/μL. The oligo sequences can be found in Supplementary Table 3.

Rooijers K., Markodimitraki C.M., Rang F.J., de Vries S.S., Chialastri A., de Luca K.L., Mooijman D., Dey S.S, & Kind J. (2019). scDam&T‐seq combines DNA adenine methyltransferase-based labeling of protein-DNA contact sites with transcriptome sequencing to analyze regulatory programs in single cells. Nature biotechnology, 37(7), 766-772.

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5 adapter

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11 protocols using 5 adapter

Yeast cell RNA extraction and 5'RACE

Vaginal Microbiome DNA Extraction and Amplification Protocol

Vaginal Microbiome 16S rDNA Profiling

Single-cell RNA-seq using CEL-Seq2

Adapter Design for Single-Cell Sequencing

Single-cell RNA-seq library preparation

RT Primer Design for Illumina Sequencing

Adapter Design for Single-Cell Sequencing

Microbiome Analysis of Fecal Samples

RT Primer Design for Illumina Sequencing

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