Single cell sequence specific amplification kit

Blastocysts were collected and immediately reverse-transcribed using Single-Cell Sequence-Specific Amplification Kit (Vazyme, Nanjing, China) according to the manufacturer’s protocol. Subsequent real-time RT-PCR was performed on a Roche Light Cycler 480 Instrument II PCR System with TB Green^® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa, Kusatsu, Japan). The expression level of each gene was normalized by Gapdh and was calculated using the 2^−△△Cq method. Five blastocysts per group were used for each reaction, and each experiment was repeated at least three times. The primer sequence is provided in Supplementary Table 1.

Based on RT-PCR amplification methods, the sorted ILC subsets were reverse- transcribed to obtain cDNA. cDNAs were synthesized with a Single Cell Sequence Specific Amplification Kit (Vazyme, Nanjing, China). First, the amplification primers of different genes were mixed to make an Assay Pool (the final concentration of each primer was 0.1 μM), and then the reaction system was configured in the Nuclease-free centrifuge tube as follows: 2x Reaction Mix 2.5ul, 0.1 uM Assay Pool 0.5ul, RT/Taq enzyme 0.1ul, Nuclease-free ddH20 up to 5.0 μl. Single-cell ILC subsets were added to the reaction system, and then the following reaction was performed on the PCR instrument: 50° for 60 min and 1 cycle, 95° for 3 min and 1 cycle, 95° for 15 s, 60° for 15 min and 14 cycles. At the end of the reaction, 20 μl of Nuclease-free ddH2O (1:5 dilution) was added to each tube, centrifuged at 3000 rpm (1000 × g) for 2 min, and subsequent qPCR reactions were performed immediately.
The qRT-PCR reactions were carried out in the Bio-Rad CFX96 Real-time PCR Detection System, using AceQ^® qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). The program was set as a two-step method, 95° for 10 s, 60° for 30 s and 40 cycles. The gene expression results were analyzed using the 2^−ΔΔCT method, and GAPDH was used as an endogenous control for mRNA expression.

To examine the expression levels of predicted DEGs between WT and MmiR-202 PGCs, the RNA of collected PGCs was reverse-transcribed with single-cell sequence-specific amplification kit to establish first the cDNA library (Vazyme, Nanjing, China). The extracted RNA of other embryos was reverse-transcribed into single-strand cDNA with primescript™ first-strand cDNA synthesis kit (Takara, Kyoto, Japan). To avoid the error caused by single reference genes in single-cell PCR validation, we used three common reference genes including ef1a, β-actin and rpl13a to normalize gene expression levels using 2^−∆∆Ct method in every PCR examination. The results were representative of more than three independent experiments in triplicate. Each independent experiment was performed in triplicate.
qPCR analysis was performed on the LightCycler480 II (Roche, Basel, Switzerland) with the chamQ universal SYBR qPCR master mix (Vazyme, Nanjing, China) as previously described [67 (link)]. Primer sequences were listed in Table A1.

Cells were directly reverse-transcribed using Single-Cell Sequence Specific Amplification Kit (Vazyme, P621-01) according to the manufacturer’s instructions. Briefly, 1000 flow-sorted MAIT cells were subjected to a 5 μl PCR reaction mix containing 2.5 μl of 2× Reaction Mix, 0.5 μl Primer Assay Pool (0.1 μM), and 0.1 μl RT/Taq enzyme. Primers used in reverse transcription-PCR (RT-PCR) are shown in Table S5. The reaction mix were quickly freezed in a −80 °C freezer for 2 min. Quantitative RT-PCR was conducted with the Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System, using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02). The expression of mRNA encoding detected genes was normalized relative to that of the mRNA encoding the internal standard RPL13a (ribosomal protein L13a).

All primers were mixed with nuclease-free water to a final concentration of 0.1 μM. cDNA was extracted from single oocytes using a single cell sequence-specific amplification kit (Vazyme, Nanjing, China), and qPCR was performed on a detection machine (Analytik Jena, Jena, Germany). The following primer sequences were used: c-Fos: forward, 5′-CTGAAGCTGACTCCTTCCCA-3′, reverse, 5′-TTGCCTTCTCTGACTGCTCA-3′; c-Jun: forward, 5′-CGCTGGAAAGCAGACACTTT-3′, reverse, 5′-TGGGTCCCTGCTTTGAGAAT-3′; Fosb: forward, 5′-CAGGAGTTGGGATGGAGGAG-3′, reverse, 5′-AACCACTCCTGGCTTATGCT-3′; Junb: forward, 5′-TCTACACCAACCTCAGCAGT-3′, reverse, 5′-ATGTGGGAGGTAGCTGATGG-3′; Jund: forward, 5′-TCCCAACTCTCCTCTGACCT-3′, reverse, 5′-TGCTGGTGTGTTTGTCTGTG-3′; Lmnb2: forward, 5′-CGTGACAAGTTCCGCAAGAT-3′, reverse, 5′-ATGTCCAGGGCCAGCTTAAT-3′; 18S: forward, 5′-ATGGCCGTTCTTAGTTGGTG-3′, reverse, 5′-CGGACATCTAAGGGCATCAC-3′. The relative expression levels were calculated via the 2^−ΔΔCt method.

Total RNA of FACS-sorted human cells was extracted using Trizol reagent (Invitrogen) and 1 μg RNA was reverse-transcribed into complementary DNA using HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) according to the manufacturer’s instructions (Vazyme). As for zebrafish, FACS-sorted ECs or HE were collected and cDNA was directly generated using Single Cell Sequence Specific Amplification Kit (Vazyme) according to the manufacturer’s instructions, diluting three to five times as templates. Quantitative PCR (qPCR) was performed on a Bio-Rad CFX96 system using ChamQ Universal SYBR qPCR Master Mix (Vazyme) and samples were run in duplicate or triplicate with R3 biological replicate pools/condition using the primers listed in Additional file 1: Table S3. Relative expression was normalized to the human actin or zebrafish actb2 housekeeping gene.

Primers pool was prepared as described previously (18 (link)). Primers used are listed in Supplementary Table S4. Individual cells were picked up into 8-strip PCR tubes with 5 µL RT-PreAmp Master Mix (1.9 µL nuclease free water, 2.5 µL Reaction Mix, and 0.1 µL RT/Taq enzyme were mixed with 0.5 µL primers pool; Single Cell Sequence Specific Amplification Kit, Vazyme, China) by special Pasteur pipettes (Brand, Germany). Eight-strip PCR tubes were immediately frozen in -80°C refrigerator for 2 min. After brief centrifugation (300 g, 4°C, 3 min), tubes were immediately moved to PCR machine following kit instructions. After preamplification, samples were diluted 100-fold with double distilled water prior to qPCR.