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Smarter ultra low input rna cdna preparation kit

Manufactured by Takara Bio
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The SMARTer Ultra Low Input RNA cDNA Preparation Kit is a laboratory equipment product designed for the conversion of small amounts of RNA into high-quality cDNA. It is suitable for use with a wide range of input RNA quantities, from picograms to hundreds of nanograms.

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Lab products found in correlation

Hiseq 2500 sequencer, by Illumina (7 mentions) Nextera xt dna library preparation kit, by Illumina (5 mentions) Nebnext ultra dna library prep kit, by Illumina (3 mentions) M220 sonicator, by Covaris (3 mentions) Acid tyrode, by Merck Group (2 mentions) Smart seq stranded kit, by Takara Bio (1 mentions) Nextseq 550, by Illumina (1 mentions) Rlt plus buffer, by Qiagen (1 mentions) Sybr green gene expression assay, by Thermo Fisher Scientific (1 mentions) Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions) Microtube 50, by Covaris (1 mentions)

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SMARTer Ultra Low RNA kit

8 protocols using smarter ultra low input rna cdna preparation kit

1

RNA-seq of Dux F2 embryos

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For embryos collected for RNA-seq (i.e., Dux F2 × F2), IVF was performed as described above except that the micro-injection steps were omitted. Late 1-cell and late 2-cell were collected at ~12 and ~30 hpi, respectively. For each biological replicate, 11–13 embryos were pooled for RNA-seq analyses. Specifically, the embryos were briefly incubated in Acid Tyrode (Millipore) to remove zona pellucida and then washed three times in 0.2% BSA/PBS prior for library construction.
RNA-seq libraries were prepared as previously described 25 . Briefly, SMARTer Ultra Low Input RNA cDNA preparation kit (Clontech, 643890) was used for reverse transcription and cDNA amplification (11 cycles). cDNA were then fragmented, adaptor-ligated and amplified using Nextera XT DNA Library Preparation Kit (Illumina) according to the manufacturer’s instructions. Single-end 100-bp sequencing was performed on a HiSeq 2500 sequencer (Illumina). The summary of the generated datasets can be found in Supplementary Table 5.
Chen Z, & Zhang Y. (2019). Loss of DUX causes minor defects in zygotic genome activation and is compatible with mouse development. Nature genetics, 51(6), 947-951.
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2

Total RNA-seq and Poly(A) RNA-seq Library Preparation

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Total RNA-seq libraries were prepared using the SMART-Seq Stranded Kit (Clontech) following the manufacturer’s instructions. Poly(A) RNA-seq libraries were prepared as previously described (56 (link)) using a SMARTer ultralow input RNA cDNA preparation kit (Clontech). A Nextera XT DNA library preparation kit (Illumina) was used for poly(A) RNA-seq cDNA fragmentation, adaptor ligation, and amplification according to the manufacturer’s instructions. The prepared RNA-seq libraries were sequenced on NextSeq 550 (Illumina) with paired-ended 75-bp reads.
Zhang C., Wang M., Li Y, & Zhang Y. (2022). Profiling and functional characterization of maternal mRNA translation during mouse maternal-to-zygotic transition. Science Advances, 8(5), eabj3967.
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3

Single-Cell RNA-Seq from Sorted Nuclei

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FANS-sorted nuclei were directly sorted to RLT plus buffer (Qiagen). Total RNA and DNA were prepared from the same samples. Total RNA samples from 400 nuclei were reverse transcribed and amplified using SMARTer Ultra Low Input RNA cDNA preparation kit (Clontech). cDNAs were then fragmented using the Covaris M220 sonicator (Covaris). The fragmented cDNAs were end-repaired, adaptor ligated and amplified using NEBNext Ultra DNA Library Prep Kit for Illumina according to the manufacturer’s instruction (New England Biolabs). Single end 100 bp sequencing was performed on a HiSeq2500 sequencer (Illumina).
Tuesta L.M., Djekidel M.N., Chen R., Lu F., Wang W., Sabatini B.L, & Zhang Y. (2019). In vivo nuclear capture and molecular profiling identifies Gmeb1 as a transcriptional regulator essential for dopamine neuron function. Nature Communications, 10, 2508.
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4

RNA-seq of Dux F2 embryos

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For embryos collected for RNA-seq (i.e., Dux F2 × F2), IVF was performed as described above except that the micro-injection steps were omitted. Late 1-cell and late 2-cell were collected at ~12 and ~30 hpi, respectively. For each biological replicate, 11–13 embryos were pooled for RNA-seq analyses. Specifically, the embryos were briefly incubated in Acid Tyrode (Millipore) to remove zona pellucida and then washed three times in 0.2% BSA/PBS prior for library construction.
RNA-seq libraries were prepared as previously described 25 . Briefly, SMARTer Ultra Low Input RNA cDNA preparation kit (Clontech, 643890) was used for reverse transcription and cDNA amplification (11 cycles). cDNA were then fragmented, adaptor-ligated and amplified using Nextera XT DNA Library Preparation Kit (Illumina) according to the manufacturer’s instructions. Single-end 100-bp sequencing was performed on a HiSeq 2500 sequencer (Illumina). The summary of the generated datasets can be found in Supplementary Table 5.
Chen Z, & Zhang Y. (2019). Loss of DUX causes minor defects in zygotic genome activation and is compatible with mouse development. Nature genetics, 51(6), 947-951.
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5

Single-Cell RNA-Seq of Blastocyst Embryos

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Six IVF or SCNT embryos of the blastocyst stage (96 hours after fertilization or activation) were directly lysed and used for cDNA synthesis using the SMARTer Ultra Low Input RNA cDNA preparation kit (Clontech, 634936). After amplification, the cDNA samples were fragmented using a Covaris M220 sonicator (Covaris). Sequencing libraries were made with the fragmented cDNA using NEBNext Ultra DNA Library Prep Kit for Illumina according to manufacturer’s instructions (New England Biolabs, E7370). Single-end 50 bp sequencing was performed on a HiSeq 2500 sequencer (Illumina). Two biological replicates were analyzed for each condition.
Matoba S., Wang H., Jiang L., Lu F., Iwabuchi K.A., Wu X., Inoue K., Yang L., Press W., Lee J.T., Ogura A., Shen L, & Zhang Y. (2018). Loss of H3K27me3 Imprinting in Somatic Cell Nuclear Transfer Embryos Disrupts Post-Implantation Development. Cell stem cell, 23(3), 343-354.e5.
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6

RNA-seq Library Preparation from Morula and ExE

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The SMARTer Ultra Low Input RNA cDNA Preparation Kit (Clontech) was used for reverse transcription and cDNA amplification following the manufacturer’s instructions. The numbers of cycles used for cDNA amplification were 10 and 13 for Dnmt3l morulae and E6.5 ExE, respectively, which yielded cDNA (0.5 to 2 ng/μl) in 20 μl. For E6.5 ExE samples, ~10 pg of cDNA was used as templates for SYBR Green gene expression assay (Invitrogen) in the ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). The threshold cycles of Dnmt3a and Dnmt3b were normalized to Gapdh using the comparative CT method. Only samples that showed minimal level of both Dnmt3a and Dnmt3b were considered as DKO. For both Dnmt3l morula and E6.5 ExE, 400 pg of cDNA obtained from the SMARTer step was used to generate RNA-seq libraries. The Nextera XT DNA Library Preparation Kit (Illumina) was used for cDNA fragmentation, adaptor ligation, and amplification (12 cycles) according to the manufacturer’s instructions. Single-ended 100-bp sequencing was performed on a HiSeq 2500 sequencer (Illumina).
Chen Z., Yin Q., Inoue A., Zhang C, & Zhang Y. (2019). Allelic H3K27me3 to allelic DNA methylation switch maintains noncanonical imprinting in extraembryonic cells. Science Advances, 5(12), eaay7246.
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7

RNA-seq Library Preparation for Embryo Samples

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RNA-seq libraries were prepared as previously described 13 (link). Briefly, reverse transcription and cDNA amplification were performed using whole embryo lysates with SMARTer Ultra Low Input RNA cDNA preparation kit (Clontech, 634890). When processing 2-cell AG, GG and α-amanitin-treated IVF embryo samples, 1 μl of 1:40,000 diluted ERCC (External RNA Controls Consortium) standard RNA (Life technologies) was added to each of the tubes at the step of cell lysis. cDNAs were then fragmented using the Covaris M220 sonicator (Covaris) with microTUBE-50 (Covaris) into average 150-160 bp fragments. The fragmented cDNAs were end-repaired, adaptor ligated and amplified using NEBNext Ultra DNA Library Prep Kit for Illumina according to the manufacturer’s instruction (New England Biolabs). Single end 100 bp sequencing was performed on a HiSeq2500 sequencer (Illumina).
Inoue A., Jiang L., Falong L., Suzuki T, & Zhang Y. (2017). Maternal H3K27me3 controls DNA methylation-independent genomic imprinting. Nature, 547(7664), 419-424.
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8

RNA-seq Library Preparation from Mouse Oocytes

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RNA-seq libraries were prepared as previously described (Matoba et al. 2014 (link)). Twenty oocytes at GO1, GO2, and FGO stages from WT and CKO female mice were used for RNA-seq library preparation. Briefly, reverse transcription and cDNA amplification were performed using SMARTer ultralow input RNA cDNA preparation kit (Clontech) following the manufacturer's instructions. Nextera XT DNA library preparation kit (Illumina) was used for cDNA fragmentation, adaptor ligation, and amplification according to the manufacturer's instructions. Single-end 100-bp sequencing was performed on a HiSeq2500 sequencer (Illumina).
Zhang C., Chen Z., Yin Q., Fu X., Li Y., Stopka T., Skoultchi A.I, & Zhang Y. (2020). The chromatin remodeler Snf2h is essential for oocyte meiotic cell cycle progression. Genes & Development, 34(3-4), 166-178.
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