Repli g wta single cell kit

The whole-genome sequencing of HRV, IAV, and hMPV was performed using samples with low Ct values (<35). RNA samples were randomly amplified using a REPLI g WTA Single Cell Kit (Qiagen, Hilden, Germany). Libraries were prepared using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) in combination with NEBNEXT Multiplex Oligos for Illumina (Dual Index Primers Set 1 and Set 2) (New England Biolabs) according to the manufacturer’s instructions. After the libraries were quantified using the NEBNext Library Quant Kit (New England Biolabs), sequencing was performed using the Miniseq High Output Kit on a Miniseq sequencer (Illumina, San Diego, CA, USA). The read data were trimmed and mapped to a reference sequence using CLC Genomics Workbench software (Qiagen). Consensus sequences were extracted as complete or nearly complete.

Sorted cells were subjected to whole transcriptome amplification using REPLI-g WTA Single Cell Kit (#150063, Qiagen) following the manufacturer’s protocol. This kit amplifies the whole transcriptome of the cells ranging from 1 to 1,000, allowing the gene expression analysis at ultra-low cell level. Amplified cDNA was analysed using qRT-PCR. Using specific primers, CTC-specific markers EpCAM, EGFR, CK-19, and Vimentin, and hematopoietic marker CD45 were detected. β-Actin/18S rRNA was used as an internal control.
We utilised three qRT-PCR strategies for the reconfirmation of sorted CTCs (*cDNA = 1:100 diluted whole transcriptome amplification (WTA) product).
The primer sequence and qRT-PCR conditions are shown in the Supplementary Material.

For low-input RNA-seq libraries, we picked up 20–50 GFP-positive germ cells at each stage by micromanipulator and transferred samples into 7 μl DPBS with a snap freeze in liquid nitrogen immediately. Reverse transcription using REPLI-g WTA Single Cell Kit (Catalog No. 150063, Qiagen, Hilden, Germany) was perfromed according to the kit’s standard protocol. The amplified cDNA was purified using 1 volume Ampure XP beads, and then fragmented to 200–400 bp by Covaris S220. NEBNext Ultra II DNA Library Prep Kit (Catalog No. E76455, NEB) was used for library construction according to manufacturer’s instruction. cDNA was end repaired and A-tailing by adding 7 μl NEBNext Ultra II End Prep Reaction Buffer and 3 μl NEBNext Ultra II End Prep Enzyme Mix. Samples were incubated in a thermal cycler at 20 °C for 30 min, 65 °C for 30 min, and finally cooled to 4 °C. Adaptor ligation was performed by adding 30 μl NEB Next Ultra II Ligation Master Mix, 1 μl NEBNext Ligation Enhancer, 0.5 μl 200 mM ATP, and 2.5 μl 25 mM cytosine methylated Illumina adapter. Sample were thoroughly mixed and incubated at 20 °C for 30 min. Adapter-ligated DNA was purified with 1 volume Ampure XP beads to discard the short fragments and adapter-self ligations, and then amplified with 6–7 PCR cycles. Sequencing was performed on Hiseq 2000 or Hiseq Xten platform (Illumina).

The sequencing results were validated by real-time PCR using nine pairs of oocytes from nine patients, respectively. The cDNA from these 18 oocytes was obtained through REPLI-g WTA Single Cell Kit (Qiagen, Germany) and was used for real-time PCR. Real-time PCR was performed with SYBR Premix Ex Taq (Tli RNaseH Plus) (Takara Bio Inc., Japan) in the LightCycler 480 II (Roche, Germany). Gene specific primers were designed by Primer Premier 5.0 Software (Premier Inc., Canada; Supplementary Table S1). The relative expression level of these genes was normalized by the housekeeping gene of GAPDH.

First, we compared two DNA extraction kits: the QIAamp DNA Microbiome kit (Qiagen, Hilden, Germany) and the QIAamp UCP Pathogen Mini kit (Qiagen) for efficacy of detection of bacterial reads. Based on the results, the QIAamp DNA Microbiome kit was used in further experiments. RNA extraction was performed using the NucleoSpin RNA Blood kit (MACHEREY-NAGEL, Düren, Germany). The extracted RNA was immediately converted to cDNA and amplified with the REPLI-g WTA Single Cell kit (Qiagen) in accordance with the manufacturer’s instructions. DNA and RNA concentrations were measured using a Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and a Qubit RNA HS assay kit (Thermo Fisher Scientific), respectively.
The Nextera XT DNA Sample Preparation Kit (Illumina, San Diego, CA, USA) was used to prepare all the libraries from extracted DNA or generated cDNA, as described above. Libraries were also prepared from distilled water as a preparation control (NTC 1-3). Library quality was analyzed using an Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA) or Agilent 2100 Bioanalyzer (Agilent Technologies). The library concentration was quantified using a Qubit dsDNA HS assay kit (Thermo Fisher Scientific). Then, libraries were sequenced on MiSeq (Illumina) with the 2 × 150 bp paired-end protocol.

cDNA was isolated from in vitro 3D CTC tumorspheres and amplified by using REPLI-g WTA Single Cell Kit (Qiagen) according to manufacturer instructions protocol. Briefly, cells were lysed followed by gDNA removal. The subsequent reverse transcription reaction was performed by using oligo dT primer to amplify polyA⁺ mRNA enrichment transcripts. The synthesized cDNA was ligated using a high-efficiency ligation mix followed by whole transcriptome amplification of cDNA with the REPLI-g SensiPhi DNA polymerase enzyme. RT-PCR were then performed by using gene specific primers (Supplementary table S4).