Agilent 2100 high sensitivity dna assay kit

The RNA of each sample was extracted and amplified based on the Smart-Seq2 protocol [22 (link)]. The PCR products (1~2 kb) were purified and recovered using Ampure XP magnific beads (BECKMAN COULTER, Shanghai, China). The quality and concentration of PCR products were determined using Agilent 2100 High Sensitivity DNA Assay Kit (Agilent Technologies, Santa Clara, CA, USA) and Qubit 3.0 Flurometer (Life Technologies, Carlsbad, CA, USA), respectively. About 20 ng cDNA of a single sample was then sheared into about 300 bp fragments using ultrasound. The fragmented cDNA was end-repaired, dA-tailed, and adaptor ligated and then subjected to further PCR amplification. The final indexed PCR products were separated using 2% agarose gel electrophoresis and recovered using Gel Extraction Kit (CWBIO, Beijing, China). The library quality was assessed using Agilent 2100 bioanalyzer, and the concentration of each sample was determined using q-PCR (effective concentration > 2 nM). Sequencing was conducted using Illumina HiSeq 2500 platform (Illumina, CA, USA) generating 150 bp paired-end reads.

The fluorescence cell counter was used again to ensure the cell number and viability of the single-cell suspensions before the assay. Single-cell transcriptome 3′ cDNA libraries were constructed using a V3.1 kit for a 10× Genomics Chromium Controller (10× Genomics Inc., Pleasanton, CA, USA), according to the instructions, with an expected capture of 10,000 cells/passage. The cDNA libraries were qualified by a Qubit^® 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and an Agilent 2100 High Sensitivity DNA Assay Kit (Agilent, Santa Clara, CA, USA). Sequencing was performed on an Illumina NovaSeq 6000 PE150 platform (Illumina, San Diego, CA, USA) at Annoroad Gene Technology Co, Ltd. (Beijing, China).

The released RNAs were reverse-transcribed, template-switched, and amplified, and then cDNA libraries were constructed by modification of the SMART-seq2 single-cell RNA-seq method [14 (link)]. The cDNA was purified and recovered using Beckman Ampure XP magnetic beads and then dissolved in an elution buffer. The cDNA was measured by an Agilent 2100 High Sensitivity DNA Assay Kit (Agilent Technologies, Santa Clara, CA, USA) to assess the length, concentration, and quality of the amplified product. The amplified cDNA was used for the library construction of single-cell transcriptomes with an insert size of about 350 bp. The 150 bp paired-end reads were generated using Illumina HiSeq 4000 sequencing platforms for further analysis according to the standard protocols provided by ANOROAD gene Technology Co., Ltd. (Beijing, China).

Smart-Seq2 method was used to amplify the single-cell sample. The reaction system for the first cDNA strand synthesis comprised the reverse transcription enzyme, buffer, template-switching oligo (TSO) primers, and oligo-dT primers with common sequence. For second-strand synthesis, the PCR amplification reagent and ISPCR primer with common sequence were added to the first-strand synthesis products. The sample cDNA was fragmented into 300 bp using a Bioruptor^®Sonication System (Diagenode Inc., Denville, NJ, USA), and then terminal repair, addition of A, ligation of sequencing adapters, and amplification were performed. The PCR amplification product, which was of 350–450 bp, was extracted. Then, the concentration was measured using a Qubit 2.0 Flurometer (Life Technologies, Carlsbad, CA, USA). The integrity of the amplified products was detected using an Agilent 2100 High-Sensitivity DNA Assay Kit (Agilent Technologies, Santa Clara, CA, USA). The libraries were sequenced on an Illumina HiSeq X Ten platform, employing 150 bp paired-end sequencing.

RNA was extracted from the 8-cell embryos fertilized and cloned using UC-MSC and fibroblasts (the three groups were labelled IVF, DFUC and DFF, respectively), and embryos were lysed using a single-cell lysis kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Subsequently, the 1st cDNA was obtained using a single-cell reverse transcription kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual and then amplified using PCR with the 1st cDNA as a template. The cDNA product was purified using Ampure XP beads and its concentration was measured with a Qubit^® 3.0 Fluorometer (Thermo, Waltham, MA, USA). The integrity and distribution of fragments were assessed using an Agilent 2100 Bioanalyzer and an Agilent 2100 High Sensitivity DNA Assay Kit (Agilent, Santa Clara, CA, USA). Library construction was performed by taking 40 ng of product from each sample of satisfactory quality and breaking it into 350 bp fragments using ultrasound. The fragments then underwent end repair, addition of base A, addition of an adapter, and PCR amplification. Finally, Novaseq S2 (Illumina, San Diego, CA, USA) was used for paired-end sequencing.