2100 bioanalyser

0.3 × 10⁶ FACS-sorted B cell subsets were washed twice with ice-cold PBS and cell pellets were snap frozen in liquid nitrogen. RNA was extracted using the RNeasy Mini Kit (Qiagen) and its quality was assessed on a 2100 Bioanalyser (Agilent). RNA integrity numbers > 7.5 of total RNA were used to generate cDNA from polyadenylated transcripts.
For Illumina sequencing, RNA was reverse transcribed using the SMART-Seq v4 ultra low input RNA kit (Takara Bio). cDNA quality was analysed on a 2100 Bioanalyser (Agilent). mRNAseq libraries were prepared using Nextera XT DNA library preparation kit (Illumina) and quantified using KAPA library quantification kit (Roche). Barcoded libraries were multiplexed and sequenced on an Illumina HiSeq 2500-RapidRun system on a 50 bp single-end mode with a coverage of 20 M reads per sample.
For Oxford Nanopore Technologies (ONT) sequencing, libraries were prepared as described previously^{90 (link),91 (link)}. Briefly, RNA was reverse transcribed using the Smart-seq2 protocol^{92 (link)}, cDNA was amplified using the KAPA HiFi Uracil+ hot start polymerase mix (Roche) and PCR products were purified using 0.6X AMPure XP beads (Beckman). Equal amounts of cDNA libraries were pooled for a total of 200 fmol and sequenced with MinION R9.4.1 flow cell using the SQK-LSK109 kit on MinKNOW (21.02.1) according to the manufacturers’ instructions.

RNA extraction of W. magna c2c maky was carried out using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The QuantiFluor RNA sample system (Promega) was used to quantify total RNA from each sample. Total RNA was qualified using Nano RNA chip on BioAnalyser 2100 (Agilent Technologies Inc.). A poly A capture was performed to purify mRNA from the total RNA. Libraries were then generated using NextFlex rapid directional RNAseq sample prep (Bioo Scientific Corporation). Libraries were quantified using dsDNA HS Assay on Quantus Fluorometer and qualified on BioAnalyser 2100 from Agilent using HS DNA chip. Sizes of fragments in the library were about 380 bp, therefore compatible with cluster generation. Paired-end sequencing with 75 bp read length was performed on NextSeq. 500 Mid Output flow cell lines from Illumina generating 130 million reads. The sequencing of two RNA samples generated 216.93 million of raw reads, 77.4% of which met the Illumina filtering criteria.

The total RNA of each sample was extracted using a RNeasy Mini Kit (Qiagen, Hilden, France) according to the manufacturer’s instructions. RNA was quantified and qualified using a QuantiFluor RNA sample system (Promega, Charbonnières, France) and a nano RNA chip on a BioAnalyser 2100 (Agilent Technologies Inc., Santa Clara, CA, United States), respectively. Illumina sequencing was performed using ProfileXpert/Viroscan3D, Lyon. Briefly, libraries were prepared from total RNA using poly(A) enrichment of the mRNA to remove ribosomal RNA. NextFlex rapid directional RNAseq sample prep (Bioo Scientific Corporation) was used to achieve the libraries. Quantification and validation of the libraries were performed using dsDNA HS Assay on a Quantus Fluorometer (Promega, Charbonnières, France) and on a BioAnalyser 2100 from Agilent using a HS DNA chip (Agilent Technologies Inc., Santa Clara, CA, United States), respectively. The library was sequenced in 75 base pair (bp) length paired end reads in NextSeq 500 Mid Output flow cell lines from Illumina (Illumina Inc, San Diego, CA, USA) generating 216.9 million raw read pairs.