GSM9475349: L141_12_dura; Mus musculus; OTHER
Firstly, we did perfusion on the mice with approximately 20 mL of precooled phosphate buffered saline (PBS) to remove blood, then excised the small intestine and rinsed the intestinal lumen with precooled PBS to remove its contents. Small intestine tissues were carefully cleaned from adipose tissue and divided into three segments equal in length named PSI (Proximal small intestine), MSI (Middle small intestine) and DSI (Distal small intestine). Each segment was cut longitudinally, starting from the most distal portion with the luminal side facing upward, the small intestine was rolled resulting in a Swiss-roll with the distal SI in the center and the proximal SI in the outer portion of the roll. The fresh tissue was then embedded in Optimal Cutting Temperature compound (OCT, Sakura Tissue-TEK) on dry ice and stored at -80 °C. The BMKMANU S3000 RNA-seq was performed with the BMKMANU S3000 Gene Expression kit (BMKMANU, ST03010). In brief, OCT blocks were cut with a pre-cooled cryostat at 10 μm thickness, and sections were transferred to fit the 6.8*6.8 mm2 oligo-barcoded capture areas on the BMK S3000 slide. The experimental slide with small intestine tissue was fixed and stained with hematoxylin and eosin (H&E) and imaged. Sequence libraries were then processed according to manufacturer's instructions. After cDNA amplification, cDNA quality inspection was performed using Agilent 2100. Before performing the complete protocol, Tissue Optimization (BMKMANU, ST03003) was performed according to manufacturer's instructions, and the fluorescent footprint was imaged using a fluorescence microscope. About thirty minutes was selected as optimal permeabilization time. In parallel, we used unfragmented cDNA as starting material and a nested PCR approach to perform targeted amplification with KAPA HiFi HotStart ReadyMix (Roche Applied Science, 07958935001). We amplified each of the three target arrays individually by performing three separate PCR reactions for each sample. We first amplified the CA-, TA-, and RA-array libraries for 9 cycles using a 1/5th of the purified cDNA, the P5_PR1 primer, and either the NGS_CA_R1, NGS_TA_R1, or NGS_RA_R1 primer. The three PCR products were purified, and each was subject to a second PCR with 9 cycles, again using the P5_PR1 primer and either the NGS_CA_R2, NGS_TA_R2, or NGS_RA_R2 primer. Each of the resulting PCR products was further purified and used to perform an indexing PCR for 9 cycles using the primers included in this kit (New England Biosciences, E6609S). We used 0.8X SPRI SELECT REAGENT beads (Beckman Coulter, B23318) to purify the PCR product from each step once except the final indexing PCR product (0.8X, twice purification). The final indexing libraries were pooled and quantified with VAHTS Library Quantification Kit for Illumina (Vazyme, NQ101), and sequenced on an Illumina NovaSeq 6000 using NovaSeq 6000 SP kits (Read 1: 85 cycles; i7 Index: 8 cycles; Read 2: 350 cycles; Illumina, MS-102-2003) with 10% PhiX sequencing control v3 (Illumina, FC-110-3001).