GSM8773090: Cartilage, Prx1Dnmt1_vivo_flox_rep2; Mus musculus; RNA-Seq
Cartilage was extracted from P5 mice. To exclude connective tissue, cartilage tussues were treated with collagenase. Total RNA was extracted from cartilage tissues using Isogene (Nippon Gene) and RNeasy spin column kit (QIAGEN), followed by treatment with DNase I (QIAGEN). BRB-seq(Alpern D et al.) was performed for preparing libraries with some following modifications. Barcoded oligo-dT based primer(5'-GCCGGTAATACGACTCACTATAGGGAGTTCTACAGTCCGACGATCNNNNNNNNNNCCCCCCCCCTTTTTTTTTTTTTTTTTTTTTTTTV -3' ; (10)N=UMI, (9)C=cell barcode) was used for single-stranded synthesis and Second Strand Synthesis Module (NEB, #E6111) was used for double-stranded cDNA synthesis. In-house MEDS-B Tn5 transposase (Picelli S et al., Sato et al.) was used for tagmentation and amplified by 10 cycles of PCR using Phusion High-Fidelity DNA Polymerase(Thermo Scientific, #M0530) and following primers(5'- AATGATACGGCGACCACCGAGATCTACACindexGTTCAGAGTTCTACAGTCCGA-3',5'-CAAGCAGAAGACGGCATACGAGATindex GTCTCGTGGGCTCGGAGATGT-3' ). 15bp of barcode read (Read1) and 81bp of insert read (Read2) were obtained on Illumina NovaSeq6000.