Nextseq 500 550 high output kit v2

sRNA Library preparation was carried out utilising QIAseq miRNA Library Preparation Kit (Qiagen, Germany, Cat no. 331505) according to manufacturer's instructions, using multiplexing adapters. Briefly, sRNA fractions were first ligated to adapters from both the 5′ and 3′ ends, reverse transcribed into cDNA using UMI‐assigning primers, and purified using magnetic beads. A universal indexing sequence to distinguish individual samples was added to each sample in the reverse transcription step. The sRNA libraries were then amplified with PCR (Eppendorf, Germany), purified and eluted into 18 μL of nuclease‐free water. The sRNA libraries were stored at −20°C until further analysis. Qubit fluorometer (Invitrogen, USA) was used to measure library concentrations, and the libraries were subsequently diluted and pooled into an equimolar mixture containing 1.8 pM per sample prior to sequencing. The sequencing of the sRNA libraries was carried out with NextSeq 500 (Illumina, USA, ), using a NextSeq 500/550 High Output Kit v. 2.5 with 75 cycles (Illumina, USA) to produce 75‐base pair single‐end reads.

Single-cell or mini-bulk RNA-seq libraries were constructed as instructed by the MATQ-seq protocol. cDNA libraries were quantified using a Qubit 3 Fluorometer with dsDNA HS Assay Kit (Thermo Fisher Scientific, Cat# Q32851). Quality check on cDNA library size (Fig. S1) was performed using Fragment Analyzer (Advanced Analytical) with HS NGS Fragment Kit (1–6000 bp) (Agilent, Cat# DNF-474-1000). Individual libraries were pooled using Nextseq500/550 High Output Kit v2.5 (Illumina, Cat# 20024906). As an optional step, pooled libraries were size-selected using Agencourt AMPure XP magnetic beads (Beckman Coulter, Cat# A63882). Pooled libraries were then subjected to scDASH treatment as illustrated in Fig. 1, followed by enrichment amplification with P5 and P7 primers. The detailed scDASH protocol can be found in Supplemental File (scDASH Protocol). Illumina libraries were sequenced using a Nextseq500/550 (Illumina) to obtain approximately 1.5 million paired sequencing reads for each library.