Smarter pico pcr cdna synthesis kit

Total RNA was extracted using an RNeasy Plus Micro kit (Qiagen), and cDNA was synthesized using the SMARTer Pico PCR cDNA Synthesis kit (Clontech). ds-cDNA was fragmented and cDNA libraries were generated using the KAPA HyperPlus Library Preparation kit (KAPA Biosystems) and FastGene Adaptor kit (Fastgene). Sequencing was performed using NextSeq500 (Illumina) with a single-read sequencing length of 60 bp, and the CLC genomic Workbench was used to analyze and visualize sequencing data. RNA sequencing data have been deposited in the DDBJ database under the accession number DRA006565.

The two cDNA libraries used for generating differentially expressed SSH libraries were synthesized from 250ng of DNase-treated total RNA isolated from FR light-induced haustoria or stems 3 d after light treatment (for each RNA isolation, material was pooled from six induced shoots) using the SMARTer Pico PCR cDNA Synthesis kit (Clontech, Mountain View, CA, USA). SSH was carried out with the PCR-Select cDNA Subtraction Kit (Clontech). The Advantage 2 PCR Kit (Clontech) was used for all PCR amplifications. After amplification, the differentially expressed cDNAs were cloned into the pGEM-T Easy vector system (Promega, Madison, WI, USA). One Shot TOP10 Chemically Competent Escherichia coli cells (Invitrogen, Carlsbad, CA, USA) were transformed with the SSH libraries and incubated on LB/Carbenicillin/X-Gal/IPTG plates at 37 °C for blue/white screening. All procedures were carried out according to the manufacturers’ instructions. Plasmid DNA was isolated from white colonies by alkaline lysis (Birnboim and Doly, 1979 (link)) and sequenced with M13F primer (5′ GTAAAACGACGGCCAGT 3′) by Sanger sequencing (Macrogen Korea, Seoul, Korea).

We prepared cDNA libraries from the venom gland for PacBio sequencing using the manufacturer’s protocol with a SMARTer Pico PCR cDNA Synthesis Kit (TAKARA Clontech) and SMRTbell Template Preparation Kit 1.0 (PacBio). We enriched longer cDNAs with a SageELF system (Sage Science, Inc). Sequencing was performed on a PacBio RS II, yielding a total of 179,143,509 reads with an average read length of 2,300 bp (Supplementary Table S4b). Most of these reads are long enough to be full-length transcripts and they were directly annotated with BLASTX against UniProt. PacBio reads are available from DRA under accession no. DRA006601.

Transcriptome analyses by PacBio reads were conducted as described in the previous our work [7 (link)]. Briefly, cDNA libraries from the venom gland for PacBio sequencing were prepared by using the manufacturer’s protocol with a SMARTer Pico PCR cDNA Synthesis Kit (TAKARA Clontech) and SMRTbell Template Preparation Kit 1.0 (PacBio). Longer cDNAs were enriched with a SageELF system (Sage Science, Inc.). Sequencing was performed on a PacBio RS II, yielding a total of 179,143,509 bps of 2300 bp average read length. N50 of PacBio reads was 5000 and not required for further assembly. Most of these reads are long enough to be full-length transcripts and directly annotated with BLASTX against UniProt. PacBio reads are publicly available from DDBJ under accession no. DRA006601.

Living sperm cell, egg cell, and embryo isolation were conducted according to the previous procedure (Zhao et al., 2014 (link)). To evaluate cell viability, embryos were stained with fluorescein diacetate (FDA). Isolated embryos were incubated in a solution containing 11% mannitol and 2 μg ml⁻¹ FDA for 15 min at room temperature and washed twice with 11% mannitol before observation (Zhao et al., 2013 (link)). Intact mRNA was directly isolated from embryos using Dynabeads mRNA DIRECT^TM Micro Kit (Life Technologies, USA), and cDNA synthesis and amplification were performed with SMARTer™ Pico PCR cDNA Synthesis Kit (Clontech, USA).

Dot1l cKO ECs from E15.5 skins and cultured Dot1l overexpression ECs were used for RT-qPCR. Total RNAs were extracted using RNeasy Plus Mini Kit (Qiagen). SMARTer Pico PCR cDNA synthesis kit (Takara) along with Advantage 2 PCR Kit (Takara) and TOPscript RT DryMix cDNA synthesis kit (Enzynomics) were used for cDNA synthesis of Dot1l cKO ECs and Dot1l overexpression ECs, respectively. RT-qPCR was performed on StepOnePlus^TM platform (Thermo Fisher Scientific) using Fast SYBR^® Green Master Mix (Thermo Fisher Scientific). Gene expression in each sample was normalized to the expression of Gapdh. The sequences for primers used for RT-qPCR are as follows: Gapdh; 5′-CAT​GGC​CTT​CCG​TGT​TCC​TA-3′, 5′-GCC​TGC​TTC​ACC​ACC​TTC​TT-3′, Dot1l; 5′-TGG​CAA​GCC​TGT​CTC​CTA​CT-3′, 5′-CTG​CTC​CTC​CCT​GAG​TTT​TG-3′, Mmp3: 5′- ACA​TGG​AGA​CTT​TGT​CCC​TTT​TG-3′, 5′-TTG​GCT​GAG​TGG​TAG​AGT​CCC-3′ and Cldn1 5′-GGG​GAC​AAC​ATC​GTG​ACC​G-3′, 5′-AGG​AGT​CGA​AGA​CTT​TGC​ACT-3′.