title: GSM9386490: Tibial osteotomy tissues, TO_4d_24h; Rattus norvegicus; RNA-Seq
library_name: GSM9386490
library_construction_protocol: 1. GEM Generation & Barcoding  Prepare Master Mix on ice, containing RT Reagent, Template Switch Oligo, Reducing Agent B, and RT Enzyme C.  Load Master Mix, cell suspension, Gel Beads, and Partitioning Oil into designated wells of Chromium Chip B.  Run the Chromium Controller to generate Gel Beads-in-Emulsion (GEMs).  Transfer GEMs to a tube strip and perform GEM-Reverse Transcription (GEM-RT) incubation (53°C for 45 min, 85°C for 5 min, then hold at 4°C).  2. Post GEM-RT Cleanup & cDNA Amplification  Add Recovery Agent to each sample and remove the lower oil phase.  Prepare Dynabeads Cleanup Mix and incubate with samples to bind cDNA.  Wash beads with 80% ethanol, elute with Elution Solution I, and transfer purified sample.  Amplify cDNA using cDNA Amplification Mix in a thermal cycler (98°C for 3 min; cycled steps: 98°C for 15s, 63°C for 20s, 72°C for 1 min; 72°C for 1 min; hold at 4°C).  Clean up amplified cDNA using SPRIselect beads (0.6X ratio), wash with ethanol, and elute with Buffer EB 3. 3' Gene Expression Library Construction  Fragmentation, End Repair & A-tailing: Incubate purified cDNA with Fragmentation Mix (32°C for 5 min, 65°C for 30 min).  Perform double-sided size selection using SPRIselect beads (0.6X and 0.8X ratios) to purify fragmented DNA.  Adaptor Ligation: Ligate adaptors using Ligation Buffer, DNA Ligase, and Adaptor Oligos (20°C for 15 min).  Clean up ligation product with SPRIselect beads (0.8X ratio).  Sample Index PCR: Amplify libraries using Sample Index PCR Mix and Chromium i7 Sample Indices (98°C for 45s; cycled steps: 98°C for 20s, 54°C for 30s, 72°C for 20s; 72°C for 1 min; hold at 4°C).  Perform a final double-sided size selection with SPRIselect beads (0.6X and 0.8X ratios) and elute the final library in Buffer EB.  4. Post-Library Quality Control  Assess library quality and size distribution using the Agilent 2100 Bioanalyzer.