Truseq rna sample preparation v2

Total RNA was extracted from Pa, Ra, Pb, and Rb samples using the TRIzol reagent (Invitrogen). For each sample, 10 μg of total RNA was subjected to mRNA purification and used for library construction using TruSeq® RNA Sample Preparation v2 (Illumina) according to the manufacturer’s instructions. We performed two biological replicates for each sample using two sequencing platforms. Paired-end 2 × 100 bp sequencing was performed on the Illumina HiSeq 2000 platform at the core facility of the Beijing Institute of Genomics, CAS, and 2 × 125 bp sequencing was performed on the Illumina HiSeq 2500 platform at BerryGenomics. High Pearson correlation coefficients (∼0.95) were observed between the biological replicates.

Total RNA was extracted using TRIzol reagent (Invitrogen, USA) and purified by poly‐T oligo–attached magnetic beads. mRNA was then fragmented into small pieces using divalent cations under elevated temperature. Then, the cleaved RNA fragments were reverse‐transcribed to create the final complementary DNA (cDNA) library in accordance with the protocol for TruSeq RNA Sample Preparation v2 (catalogs RS‐122–2001 and RS‐122–2002, Illumina, USA). The average insert size for the paired‐end libraries was 300 ± 50 base pairs (bp). Then, we performed the paired‐end sequencing on an Illumina X10 (San Diego, CA, USA) at the LC Biotechnology Co. (Hangzhou, Zhejiang, China).

The sequencing libraries were made with TruSeq RNA Sample Preparation v2 (Illumina) according to the manufacturer’s instructions. The samples were sequenced at different times, all on Illumina sequencing machines (HiSeq2500 or NovaSeq6000) with paired-end sequencing and a read length of 126 and 151 nucleotides respectively. All sequencing data were mapped to D. melanogaster genome version 6.33 using STAR version 2.7.0e and default parameters. For each gene and each time point, reads were counted using HTSeq version 0.9.1. The BRIC-seq and RNA-seq data reported in this paper have been deposited in the European Nucleotide Archive with accession number PRJEB15335.

Total RNA was purified from individual biological replicates (four for both cercariae and schistosomula) using TRIzol, and residual genomic DNA removed (DNA-free kit, Invitrogen) following the manufacturer’s instructions. The integrity and quality of total RNA were determined using a Bioanalyzer 2100 (Agilent) and Qubit RNA BR assay kit (Invitrogen). Messenger RNA (mRNA) was purified, and short-insert (330 bp) complementary DNA (cDNA) libraries constructed and barcoded according to the manufacturer’s instructions (TruSeq RNA Sample Preparation v.2, Illumina). All cDNA library was paired-end sequenced (2 × 211 base reads) on a single line using the HiSeq 2500 platform (Illumina).

Illumina TruSeq RNA sample preparation v2 (Illumina, United States) was used to prepare RNA samples for sequencing on a next-generation sequencing platform. Following the manufacturer’s directions, total RNA was extracted from HGMCs using Redzol reagent (SBS Genetech, Beijing, China). FASTQC was used to evaluate read quality, and Seqtk (v1.3-r106) was used to filter it. After that, using HISAT2 with default settings, clean reads were mapped to the human reference genome version 38 (GRCh38/hg38). The expression of transcripts was then determined using the StringTie programme. The mRNAs with |Fold Change| ≥ 2 and p-value < 0.05 were considered significantly differentially expressed (Robinson et al., 2010 (link); Sui et al., 2022 (link)).

Total RNAs were extracted from these samples using the RNA simple Total RNA Kit (Tiangen, China) according to the manufacturer's instructions and were then treated with DNase I to remove potential genomic DNA contamination. After that, the RNAs were examined by gel electrophoresis and quantified with NanoDrop. Integrity of the quantified RNA samples was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The cDNA libraries were constructed using TruSeq RNA Sample Preparation V2 (Illumina, USA) according to the manufacturer's instructions. After quality control to detect fragment size and concentration, the libraries were sequenced using the Illumina HiSeq 2500 system. All sequence data were uploaded to the NCBI SRA database with the SRA accession number PRJNA639011.

Root explants derived from wild type (Col-0) and ldl3-1 seedlings were collected on days 0 and 14 on CIM (C0, C14), and days 1 and 7 on SIM (C14S1, C14S7). Total RNA was isolated from the collected explants using the PureLink Plant RNA Reagent (Thermo Fisher Scientific). The integrity of purified RNA was assessed using a 2100 Bioanalyzer (Agilent). A total of 1000 ng RNA was used to construct a transcriptome library with TruSeq RNA Sample Preparation v.2 (Illumina). Libraries were pooled and 36–86 bp single-read sequences were obtained with a NextSeq 500 sequencer (Illumina). Three independent biological replicates were analyzed for each genotype.