Rna clean concentrator 5

The oligonucleotides used as templates for crRNA transcription and the primers used for DNA amplification were synthesized by GenScript (Supplementary Table 5). crRNA was prepared with in vitro transcription. To prepare the templates for crRNA synthesis, two paired primers containing a T7 priming site were synthesized and annealed in Taq DNA Polymerase PCR Buffer (Thermo Fisher Scientific). The crRNAs were then transcribed with the HiScribe™ T7 Quick High Yield RNA Synthesis Kit (NEB) and purified with RNA Clean & Concentrator^TM-5 (Zymo Research). The resulting crRNA was quantified with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Throughout the experiments, RNase free materials and conditions were applied.

Five adult F2-females immobilized at 0.2 µg/L (S from family group 1) and five adult F2-females not visibly affected at 1 µg/L (R from family group 2) in addition to female parasites from the P0 and 2013 Ls A (n = 4 and n = 3) and Ls V (n = 5 and n = 7) groups were prepared for RNAseq analysis.
Total RNA was extracted from individual adult females using a Trizol protocol combined with RNeasy Mini kit for animal tissues (Qiagen, CA, USA). Lice tissues were disrupted and homogenized in 1 ml Trizol using TissueLyser MM 301 (Qiagen Retsch) and one stainless steel bead of 5 mm diameter (Qiagen). After mixing with 0.2 ml of chloroform and a centrifugation step, the aqueous phase was transferred to a new vial and mixed with one volume of 70% ethanol. Total RNA was then isolated with RNeasy spin columns following manufacturer’s protocol. Genomic DNA was removed from the extracted RNA with Turbo DNA-free TM kit (TURBO™ DNase Treatment and Removal Reagents, Ambion, Life Technologies). Subsequently, the RNA was cleaned and concentrated with RNA Clean & ConcentratorTM-5 (Zymo Research). The RNA was quantified with ND-100 Spectrophotometer (Thermo Fisher Scientific, DE, USA) and the quality was checked with a 2100 Bioanalyzer instrument (Agilent Technologies) and the Agilent RNA 6000 Nano kit.

Seeds of S. vaccaria L. ‘pink beauty’ (johnnyseeds.com) were sown on moist filter paper in the dark at room temperature until germination. Plates of seeds were then placed in a growth room (20 °C, 12 h light) for 3 days. Seedlings were transferred to a growth tray filled with hydroponics solution^{48 (link)}. Plants were grown in a growth chamber at 50% humidity, 14-h photoperiod, and 18 °C for 5 weeks. Methyl Jasmonate (MeJA) (MillliporeSigma, catalog number 392707) was added to hydroponic solution at 50 or 100 μM. Leaves and flowers were collected from four individual plants of control or MeJA group after 4 to 72 h of treatment. Tissues were immediately frozen in liquid nitrogen and stored at −80 °C until further use.
Total RNA was extracted using TRIzol RNA Isolation Reagent (Thermo Fisher Scientific, Waltham, MA) according to the vendor’s manual. DNA was digested and removed with a TURBO DNA-free^TM Kit (Thermo Fisher Scientific). RNA was purified and concentrated by using an RNA Clean & Concentrator^TM -5 (ZYMO RESEARCH). RNA integrity was measured on a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA). Individual RNA samples with RNA integrity number (RIN) > 7.0 were quantified and aliquoted for library preparation and sequencing as described below.

The other half of the total cDNA (10 μl cDNA obtained in the nested RT-PCR step) was used to construct the NGS libraries. Preliminary PCR was carried out with KAPA HiFi HotStart ReadyMix. PCR amplicons were incubated with Exo I (NEB, M0568) at 37 °C for 15 min to eliminate ssDNA in the reaction volume and were then purified by VAHTS DNA Clean Beads (Vazyme, N411-01) at a 1:1 ratio.
A total of 140 ng of amplified products was fragmented by dsDNA Fragmentase (NEB, M0348), and Illumina sequencing libraries were constructed using the NEBNext Ultra II end repair-dA tailing module (NEB, E7546) and ligation module (NEB, E7595). Seven to nine cycles were performed for the final PCR amplification, depending on the DNA fragments used as input. Finally, the region from 200 bp to 700 bp was excised and recovered from 4% Nusieve 3:1 agarose (LONZA, 50090) by a Zymoclean Gel DNA Recovery Kit (Zymo, D4008). During the process of library construction, DNA purification was carried out by VAHTS DNA Clean Beads, and RNA or cDNA fragments longer than ~100 bp were purified by RNA Clean & Concentrator^TM-5 (Zymo, R1015) or Oligo Clean & Concentrator (Zymo, D4061) kits according to the specifications.

About 1.5 × 10⁶ cells were seeded in 100 mm tissue culture dishes 24 h prior to the addition of AFA 10 μM, GANT 20 μM, AFA 10 + GANT 20 μM or DMSO. After 24 h, total mRNA was extracted using TRizol (Invitrogen-Thermo Fisher Scientific, Waltham, CA, USA) and RNA Clean & ConcentratorTM-5 (R1014, Zymo Research, Irvine, CA, USA) according to manufacturer’s instructions. cDNA synthesis was performed using the High-Capacity cDNA reverse transcription kit (BIO-65054, Meridian Bioscience, Cincinnati, OH, USA). Quantitative real-time PCR analysis of specific mRNA levels (GLI1, GLI2, Ptch1) was performed on cDNAs employing TaqMan gene expression assay (Applied Biosystem, Thermo Fisher Scientific) and using the ViiATM7 Real-Time PCR System (Applied Biosystem, Thermo Fisher Scientific). Primers for gene expression were Hs00171790_m1 GLI1, Hs01119974_m1 GLI2, Hs00181117_m1 Ptch1 (Applied Biosystem, Thermo Fisher Scientific). Experiments were biologically replicated at least three times, and all of them were performed with three technical replicates. Relative mRNA expression was normalized on the mean of expression of four housekeeping genes (GAPDH, TBP, HPRT and β-Actin) and it was calculated using the ΔΔCt method as in Spiombi et al. [53 (link)].

Total RNA was isolated from cell lines and tissues using TRIzol (Invitrogen) followed by 30 min of RNase-free DNaseI treatment (NEB) at 37°C and RNA Clean & Concentrator^TM-5 (Zymo Research). 2 μg of total RNA was used to generate cDNA using Superscript III Reverse Transcriptase (Invitrogen) with random hexamers (Invitrogen) according to manufacturer's instructions.
Resulting cDNAs were analyzed of transcript-specific expression through quantitative reverse-transcript PCR (qRT-PCR) using Power SYBR Green PCR master mix (Applied Biosystems) with custom-designed primer sets (Supplementary Table 6) purchased from Integrated DNA Technology. Relative expression was determined by normalizing the expression of all genes of interest to either human or mouse Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) expression (ΔCt) as described [56 (link)].

The preparation of crRNA proceeded in three steps. The transcription templates for crRNA preparation were amplified by the PCR process, with the primers listed in Table 2. Then, the transcription process was performed at 37 °C overnight using the T7 High Yield Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). Finally, the transcript products were purified using the RNA Clean & ConcentratorTM-5 (Zymo Research, Irvine, CA, USA) and quantified with NanoDrop 2000C (Thermo Fisher Scientific, Waltham, MA, USA).