Hs ngs fragment kit 1 6000bp

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.

Single cells were collected from P2 Fgfr3-tdTomato/Sox2-GFP and Lfng-GFP mice’ cochlear ducts and sorted into 96-well plates pre-filled with lysis buffer (2U/μL RNase inhibitor, 0.2% (vol/vol) Triton X-100, 2.5 μM modified Oligo-dT primer (5′- AAG CAG TGG TAT CAA CGC AGA GTA CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TVN - 3′). Reverse transcription was performed with SMARTScribe Reverse Transcriptase (Clontech) followed by cDNA synthesis according to the SmartSeq2 protocol (Picelli et al., 2014 (link)). Synthesized cDNA was purified with AMPure XP beads (Beckman Coulter) and then quantified and assessed for size distribution with the HS NGS Fragment Kit (1–6000bp) (Agilent) and a fragment analyzer (Advanced Analytical Technologies, Inc.). Samples with more than 0.05 ng/μL of cDNA were selected using PACTAN software (Sinha et al., 2017 (link)). 98.7% of the samples passed the criteria and were concentration-normalized and tagmented with the Nextra XT (Illumina) sample preparation system, adding cell-specific barcodes to the fragments. The samples were amplified by PCR and purified with AMPure XP beads. Purified cDNA library was quality assessed with a Bioanalyzer 2100 (Agilent). Sequencing was performed on an Illumina NextSeq 500 high output flow cell configuration with paired-end sequencing of 150 bp.