GSM9709539: SHAM,DAY7,SAMPLE1; Mus musculus; RNA-Seq
After successful establishment of the SNI model, mice were randomly selected for tissue sampling. The whole fresh hippocampus tissues were placed in sterile petri dishes and rinsed with 10 mL of 4 °C pre-chilled DPBS, repeated 1-2 times to remove residual tissue protection solution. The rinsed tissues were placed in a new petri dish. After adding type II collagenase working solution, the tissues were cut into small pieces of about 1-2 mm3. The cut tissues were transferred to a 50 mL centrifuge tube containing 5 mL of digestion solution and placed in a 37 °C water bath for shaking and digestion. After digestion, the digest was filtered through a pre-wetted 70 μm cell sieve. Thereafter, the filtrate was collected into a new 50 mL centrifuge tube. After washing with 10 mL of DPBS containing 2% FBS, the filtrate was collected into the same centrifuge tube. Following centrifugation at 900 g for 30 min at room temperature, the supernatant was discarded. The pellet was added to 5 mL of DPBS containing 2% FBS, and the cells were resuspended by gentle pipetting and mixing. After centrifugation and the supernatant discarded, 3 mL of pre-chilled red blood cell lysate was added to the cell pellet and incubated at room temperature for 3 min. After discarding the supernatant, the pellet was added to 5 mL of pre-chilled DPBS containing 2% FBS. The cell suspension was stained by trypan blue and was counted using Countess® II Cell Counting. If the cell viability was more than 80%, 10× Genomics sample processing was carried out. The prepared cell suspension, 10× barcode gel magnetic beads and oil were added to different chambers of Chromium Chip B respectively, and Gel Beads-Emulsion (GEM) was formed through the microfluidic cross system. The GEM was transferred to the PCR machine for reverse transcription, with 30 nt oligo-dT reverse transcription primers on the gel magnetic beads. The poly-A RNA in cells was reverse transcribed into first strand cDNA with barcode and unique molecular identifiers (UMI) information. After magnetic bead purification of first-strand cDNA, the cDNA was amplified by RT-PCR. Afterwards, amplified cDNA was digested and fragmented, and the optimal fragment was screened with magnetic beads. The cDNA library containing P5 and P7 adapters was prepared by end-repairing, adding A, and connecting Read 2 sequencing primers with adapters. Following purification, the concentration of cDNA was measured using Qubit and fragment size was measured using Agilent 2100.The libraries were sequenced on Novaseq 6000.