Truseq stranded total rna lt sample prep kit

Total RNA was isolated from fungal and bacterial cells that were co-cultured for 8 h in six-well plates. For bacterial RNA isolation, the cells were disrupted using glass beads. For fungal RNA isolation, cells were frozen and homogenized using mortar and pestle, and then the total RNA was extracted using an RNA isolation kit (RNeasy Mini Kit; QIAGEN). Ribosomal RNA was depleted using Ribo-ZERO magnetic kit (Epicentre). The transcripts were fragmented and used as templates to generate strand-specific cDNA libraries by TruSeq Stranded Total RNA LT Sample Prep kit (Illumina). Each sample was sequenced using 100-bp paired-end reads on an Illumina HiSeq 2500 instrument. Macrogen Inc. supported library preparation, sequencing and partial data analysis. The reads were mapped to reference genomes of B. subtilis NCIB3610 (CP020102.1) or A. nidulans TN02A3 (GCA_000149205.1) with Bowtie 2 aligner. Read count per gene was extracted from known gene annotations with HTSeq program. After log₂ transformation of RPKM+1 and quantile normalization, differentially expressed genes were selected on conditions of log₂ > 2 in expression level.

Total RNA of N-replete and N-deplete cells on day 3 and day 5 were used for RNA-seq as they correspond to late-exponential and stationary phases of cells grown under N-deplete conditions, compared to N-replete cells which are in exponential phase. RNA integrity was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies) using the RNA 6000 Nano Kit (Cat. no. 5067–1511; Agilent Technologies). Construction of cDNA was performed with approximately 2 μg of RNA using a TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina) following the manufacturer’s procedures (first and second strand cDNA synthesis, 3′ end adenylation, adapter ligation, DNA fragment enrichment of ~300 bp in length). Ribo-Zero rRNA Removal Kit for Plant (Illumina) was used to reduce ribosomal RNA amount in each sample. The quality of constructed cDNA libraries and size of DNA fragments were validated on the Bioanalyzer with Agilent DNA 1000 kit (Cat. no. 5067–1505; Agilent Technologies), before being quantified by qPCR with the KAPA Library Quantification Kit for Illumina platforms (Cat. no. KK4824; Kapa Biosystems).

RNA-seq libraries were generated with total RNA extracted from tissues by using the commercial kit of Illumina TruSeq Stranded Total RNA LT Sample Prep Kit. We performed paired-end sequencing, generating a 101 nucleotides sequencing read for each end. Sequencing reads were aligned to the human reference genome by the STAR software (version: 2.6.0c)^{59 (link)}, using the primary assembly and gene annotation obtained from GENCODE (GRCh38 ‘primary_assembly’ and ‘comprehensive gene annotation (regions: CHR)’ version 27). The potential PCR duplicates were marked by the Picard MarkDuplicates (version: 2.6.0). The numbers of reads assigned to gene bodies were counted in a strand-specific way by using FeatureCounts (version: subread 1.6.2) with the command-line options of ‘-g gene_id -C -s2 -p’^{60 (link)}.

RNA was extracted using the Omega Biotek E.Z.N.A. Plant RNA kit according to the manufacturer’s protocol. RNA quality was examined on a 1% agarose gel and RNA concentration was quantified using the Qubit RNA HS assay kit (Invitrogen, USA). 2μg of total RNA was used to construct stranded RNAseq library using the Illumina TruSeq stranded total RNA LT sample prep kit (RS-122-2401 and RS-122-2402). Multiplexed libraries were pooled and sequenced on HiSeq4000 using paired end 150nt mode.

RNA isolation was performed as for PHDFs and MEFs. Cells were lysed with 27-gauge, 1/2-in. needles and then homogenized with QIAshredder columns (Qiagen). Total RNA from triplicate experiments was purified with the RNeasy Plus kit (Qiagen). The quality of purified total RNA samples was determined with an Agilent 2100 Bioanalyzer, and only samples with an RNA integrity number (RIN) of 9 or higher were used. RNA concentration was measured with a Qubit fluorimeter prior to library prep. Four micrograms of total DNase-treated RNA was run through the TruSeq Stranded Total RNA LT sample prep kit from Illumina as previously described (18 (link)). Samples were quantified by Qubit before being normalized, pooled, and then sequenced on the Illumina HiSeq 2500 sequencer with SBS v3 reagents. Each sample was sequenced at a depth of at least 25 million 50-nucleotide single-end reads.