title: GSM8630813: old-2, 21M; Mus musculus; ChIP-Seq
library_construction_protocol: Gastrocnemius muscle tissues were washed twice in cold PBS buffer and cross-linked with 1% formaldehyde for 10 minutes at room temperature before quenched by addition of glycine  (125 mmol/L). Afterwards, samples were resuspended in lysis buffer  (50mM Tris-HCl  (pH 8.0), 10mM EDTA, 1%SDS, 1×protease inhibitor cocktail) and sonicated to obtain soluble sheared chromatin  (average DNA length of 200–500 bp). 100μL chromatin was immunoprecipitated by 10 μg anti-TRα antibodies  (ab53729， abcam) at 4 ℃ overnight and further incubated with 30 μL of protein beads for 3h. The beads were next washed extensively and bound material was then eluted from the beads in 300μL of elution buffer  (100 mM NaHCO3, 1%SDS). Immunocomplexes were then treated with RNase A  (8μg/mL) during 6h at 65℃ and decrosslinked with proteinase K  (345μg/mL) overnight at 45℃. Immunoprecipitated DNA was used to construct sequencing libraries following the protocol provided by the I NEXTFLEX® ChIP-seq Library Prep Kit for Illumina® Sequencing（NOVA-5143-02， Bioo Scientific） and sequenced on Illumina Novaseq 6000 with PE 150 method.