Mrna seq sample prep kit

RNA-seq libraries were prepared from parasite stages in accordance with the RNA-seq protocols of the Illumina mRNA-Seq Sample Prep kit and the Illumina TruSeq kit. The reads were analyzed as previously described (64 (link)). Genes that had blast scores of <30% of the maximum possible score (self-blast) in other nematodes with E values of >1E-05 were considered unique. The normalized transcriptome data were analyzed in JMP Genomics (SAS Inc., Cary, NC) for general assessment of distribution analyses, correlations, principal-component analyses, ANOVA, hierarchical clustering and heat map generation, and parallel coordinate plots.

MDA231 cells were treated with (i) vehicle, (ii) 5 μM MK-4827 (PARPi), (iii) 2.5 μM AZD-1775 (Wee1i) and (iv) combination of MK-4827 and AZD-1775 for 48 h and cultured in drug free media for an additional 48 h, harvested and total RNA from each sample was isolated using a RNeasy Kit with DNase treatment (#74104 and #79254, Qiagen, Germantown, MD, USA). RNA samples were submitted to the Sequencing and Microarray Core Facility at MD Anderson Cancer Center. mRNAs were enriched by Poly(A) selection, and the libraries were prepared by mRNA-Seq Sample Prep Kit (Illumina, Foster City, CA, USA) following the manufacturer’s instructions. Pair-end 76-bp sequencing was performed by Illumina HiSeq 2000. FASTQ sequence files were obtained, and the RNA-seq reads were aligned to the human reference genome. Functional analysis of the differentially expressed transcripts was performed using gene set enrichment analysis (GSEA) as described [73 (link)].

HeLa cells transfected with expression constructs were washed with phosphate-buffered saline (PBS) (−) five times. Total RNA purification and immunoprecipitation (IP) were performed with the RiboCluster Profiler RIP-Assay Kit #RN1001 (MBL International Corporation, Woburn, MA, USA). Immunoprecipitation was carried out using anti-GFP antibody (Clonetech Laboratories, Mountain View, CA, USA) or normal rabbit immunoglobulin G (IgG) as a control. Total RNA was purified with DNase (TURBO™ DNase (0.88 U) (Invitrogen)) and fragmented using mRNA-Seq Sample Prep Kit (Illumina, San Diego, CA, USA). RNA sized 200–400 nt was excised from the acrylamide gel and subject to library preparation. Cluster amplification and single-end sequencing was performed using the Illumina TruSeq SBS Kit v5-GA #FC-104-5001 and Illumina Genome Analyzer GAIIx according to the manufacturer’s protocol (read length: 36 nt). The sequence data was first converted to qseq format by CASAVA v1.8.2 (Illumina), and further converted to FASTQ format using the qseq2fastq converter provided by Kris Popendorf [12 ], and reads containing any base with a Phred quality score of less than 20 were filtered out using FASTX toolkit 0.0.13 [13 ] and custom python code to remove reads containing N’s and homo-polymers consisting solely of one type of nucleotide.

Bacteria were lysed by constantly grinding in the presence of liquid nitrogen and total RNA was extracted using TRIzol reagent (Invitrogen). RNA was fully treated with RNase-free DNase I (Takara) to prevent contamination of trace genomic DNA [8 (link), 42 (link)]. The RNA quality was evaluated by the BioAnalyzer 2100 system (Agilent) and ribosomal RNAs were removed with the RiboZero rRNA removal kit (Epicenter) for gram-positive organisms prior to sequencing analysis. After deprivation of the rRNA, RNA was fragmented and used as a template for a randomly primed PCR.
Strand-specific cDNA libraries were prepared by standard techniques for subsequent Illumina sequencing using the mRNA-seq Sample Prep kit (Illumina). The concentration of the cDNA library was detected by Qubit®2.0 Fluorometer and verified for appropriate fragment size (200–300 bp) on a BioAnalyzer 2100 system (Agilent). Samples were amplified onto flowcells using an Illumina cBot and sequenced on an Illumina HiSeq 2500 for 51 cycles according to manufacturer protocols. Raw sequencing data was processed using the data collection software provided by Illumina [43 (link)].

More than 40 million single-end reads 50 bp long were obtained for each sample of cortical RNA, by Illumina nonstranded sequencing on an Illumina GAIIx instrument at the Genoanalitika Lab, Moscow, Russia [http://www.genoanalytica.ru/, (accessed on 1 September 2021)] in accordance with standard Illumina protocols (mRNA-Seq Sample Prep Kit). Briefly, polyadenylated mRNA was purified from total RNA using Sera-Mag Magnetic Oligo (dT) beads and then broken into small fragments by means of divalent cations and heating. Using a reverse transcriptase and random primers, we synthesized the first- and second-strand cDNAs. The cDNA was processed in an end repair reaction with T4 DNA polymerase and Klenow DNA polymerase to blunt the termini. An “A” base was then added to the 3′ end of the blunt phosphorylated DNA fragments, and an Illumina adaptor with a single T overhang at its 3′ end was then ligated to the end of the DNA fragment, for hybridization in a single-read flow cell. After that, a size range of cDNA templates was selected, and these fragments were amplified on a cluster station using the Single-Read Cluster Generation Kit v2. Sequencing-by-synthesis (SBS) at 50-nucleotide length was performed by means of SBS v4 reagents on a Genome Analyzer IIx running the SCS2.8 software (Illumina).