Transcriptional profiling of L4-L6 DRGs in Twik-1 knockout mice following chronic constriction injury

To identify the molecular mechanisms of reduced pain response in Twik-1 knockout mice, we performed bulk RNA-sequencing on lumbar DRGs from WT and Twik-1 knockout mice. Thirty days after sciatic nerve injury, the ipsilateral L4-L6 DRGs were manually sectioned into ~150 µm slices using a razor blade. RNAs were extracted from the sliced DRGs using PicoPure™ RNA Isolation Kit. RNA libraries were constructed using the SMART-Seq v4 Ultra Low Input RNA Kit. High-quality libraries were pooled and sequenced on the Illumina NovaSeq 6000 platform with 150 bp paired-end reads. Image analysis was performed using the NovaSeq 6000 Control Software (v1.8.2), and base calling data were demultiplexed using bcl2fastq (v2.20.0.422), generating FASTQ files. Raw sequencing reads were aligned to the Mus musculus reference genome (GRCm39) using STAR aligner (version 2.7.11a). The genome index was built from the primary assembly FASTA file (Mus_musculus.GRCm39.dna.primary_assembly.fa.gz) and the corresponding gene annotation file (Mus_musculus.GRCm39.113.gtf.gz), ensuring accurate mapping across exon–intron boundaries. Resulting SAM/BAM files were then processed, sorted, and indexed with Samtools (version 1.18). Gene-level counts were generated using htseq-count (version 2.0.3) with the corresponding GTF annotation file.