Total rna nano chip

RNA was extracted from frozen, pulverized forebrains using TRIzol reagent (Invitrogen). Extracted RNA was cleaned using RNeasy mini extraction kit with on-column DNase treatment (Qiagen). RNA quality was quantified with the Qubit RNA HS assay (Invitrogen). RNA quality was checked on an Agilent Bioanalyzer 2100 with the Eukaryote Total RNA Nano chip (Additional File 1: Table S1). Total RNA (1 µg) was used for sequencing library preparation using the Illumina TruSeq RNA library prep with polyA purification (Additional File 1: Table S1). Libraries were loaded at equimolar quantities and sequenced on paired-end, 75 bp runs on the Nextseq 500 (Illumina) with a goal of attaining a yield of 30–50 Mb of sequence per sample. RNA extraction and sequencing was performed with an aim to reduce batch effects.

Total RNA was extracted from 1-ml aliquots of the communities with the addition of S. mutans, described in the previous section, using the RNEasy Power microbiome kit (Qiagen, Germantown, MD, USA) as per the manufacturer's instructions. rRNA was depleted using the CRISPRclean Pan Bacterial rRNA Depletion kit (Jumpcode Genomics, San Diego, CA, USA). Short read libraries were prepared using the NEBNext Ultra II Total RNA Library Prep kit (New England Biolabs, Ipswich, MA, USA) and sequenced for 300 cycles on a NovaSeq 6000 S2 (Illumina, San Diego, CA, USA). Integrity of the mRNA and libraries was verified using a Total RNA Nano chip (Agilent, Santa Clara, CA, USA) on a Bioanalyzer (Agilent, Santa Clara, CA, USA). Relative abundances and the predicted read counts of taxa were obtained using MetaPhlAn3 [37 (link)]. Beta diversity using the Robust Aitchison metric and PCA analysis with feature loadings was performed using the DEICODE plugin for QIIME2 on the predicted read counts. Functional annotation of the mRNA was performed using HumanN3 [37 (link)]. RNA-seq reads were mapped to the B04Sm5 genome using Burrows-Wheeler Aligner (BWA) MEM [38 (link)]. The number of reads mapping to each genomic feature (e.g., gene) was determined using the featureCounts tool from the Subread software package [39 (link)]. Differential abundance analysis was performed using DESeq2 [24 (link)].

Total RNA was isolated from, at least, three different samples of ascocarps of each of the desert truffles T. claveryi, Terfezia arenaria (Moris) Trappe and Picoa lefebvrei (Pat.) Maire, mycelium of Tirmania nivea (Desf.) Trappe or yeast using spin columns (RNeasy Plant Mini Kit, Qiagen) according to the manufacturer’s instructions. In the case of T. claveryi, the CTAB protocol (Chang et al., 1993 (link)) or the phenol and chloroform extraction (Kay et al., 1987 (link)) were also used for RNA extraction, both in ascocarps and mycelium.
For quality and integrity analysis, T. claveryi, T. arenaria, T. nivea, and P. lefebvrei RNAs were electrophoretically separated in agarose gel or with an Agilent 2100 Bioanalyzer, using a 2100 expert_Eukaryote total RNA Nano Chip. When RNA was heat denatured prior to separation, RNA was incubated at 95°C for 2 min.

Samples from 12 individual mice (uninfected Reg1^+/+ n = 3, uninfected Reg1^+/− n = 3, KP-infected Reg1^+/+ n = 4, KP-infected Reg1^+/− n = 4) were used. Mice were infected with KP by oropharyngeal aspiration. After 24 h, alveolar macrophages were stained as described above and sorted by flow cytometry with a purity of 99%. Cells were placed into RLT-plus (Qiagen, Valencia, CA) and total RNA extracted using RNeasy MiniKits (Qiagen). RNA was quantitated using Nanodrop and integrity determined with a total RNA Nano Chip (Agilent Technologies). Single-stranded total RNA-seq libraries were sequenced with an Illumina Nextseq500 sequencer with a depth of 25 million reads per sample (75 bp single-end) at the University of Pittsburgh Health Sciences Sequencing Core. Fastq files with high quality reads (phred score >30) were uploaded to the CLC Genomics Workbench (Qiagen) and reads aligned to the mouse reference genome. Transcript counts and differential expression analyses were carried out using the CLC Genomics Workbench. RNAseq data were deposited to Sequence Read Archive (SRA), BioProject ID: PRJNA789160.

Total mRNA was extracted from four parental SK-MEL-239 and four vemurafenib-resistant SK-MEL-239 RNA biological replicates, using RNeasy Mini Kit (Qiagen, #74104, Lot 163028041, Hilden, Germany) according to the manufacturer’s protocol, and DNA was digested with DNA-free™ DNA Removal Kit (Invitrogen, #AM1906, Lot 00626944, Waltham, MA, USA). RNA integrity was assessed on 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) using a Eukaryote Total RNA Nano Chip (version 2.6), with samples’ RIN value range ranging from 8.9 to 10. PolyA selection was performed using NEB Next^® Ultra ™ RNA Library Prep Kit (New England Biolabs, # E7530L, Ipswich, MA, USA) and the sequencing libraries (250~300 bp insert cDNA library) were generated with a proprietary methodology developed by Novogene, China. We performed 150 bp paired-end sequencing on Illumina NovaSeq 6000 platform (Novogene, Beijing, China).