GSM9113549: Ribo-Seq Eif5a SKO round spermatids_rep3; Mus musculus; OTHER
Round sperm samples were lysed in ice-cold lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml CHX, 1% Triton X-100, 25 U/ml Turbo DNase) for 4 hours on ice. Lysates were clarified at 20,000 ×g for 10 minutes at 4°C, treated with RNase I (1 µl of 100 U/µl) at room temperature for 45 minutes, and the reaction terminated with 10 μl SUPERase•In. Samples were layered onto a 700 µl sucrose cushion (1 M sucrose, 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml CHX, 20 U/ml SUPERase•In) and centrifuged at 260,000 ×g, 4°C for 4 hours. Pellets were resuspended in 50 µl precipitate buffer, and RPFs were extracted using TRIzol and chloroform. RPFs were precipitated with isopropanol and glycogen at -20°C and resuspended in nuclease-free water. RPFs were separated on a 15% TBE-urea polyacrylamide gel (200 V, 65 min), and RNA was extracted from gel slices using RNA extraction buffer, precipitated, and resuspended in nuclease-free water. Sequencing libraries were prepared using the CATS Small RNA-seq Kit or D-Plex Small RNA-seq Kit, followed by purification with AMPure XP. Barcoded libraries were pooled and sequenced on Illumina platforms, generating 150 bp paired-end reads. Round sperm samples were lysed in ice-cold lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml CHX, 1% Triton X-100, 25 U/ml Turbo DNase) for 4 hours on ice. Lysates were clarified at 20,000 ×g for 10 minutes at 4°C, treated with RNase I (1 µl of 100 U/µl) at room temperature for 45 minutes, and the reaction terminated with 10 μl SUPERase•In. Samples were layered onto a 700 µl sucrose cushion (1 M sucrose, 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml CHX, 20 U/ml SUPERase•In) and centrifuged at 260,000 ×g, 4°C for 4 hours. Pellets were resuspended in 50 µl precipitate buffer, and RPFs were extracted using TRIzol and chloroform. RPFs were precipitated with isopropanol and glycogen at -20°C and resuspended in nuclease-free water. RPFs were separated on a 15% TBE-urea polyacrylamide gel (200 V, 65 min), and RNA was extracted from gel slices using RNA extraction buffer, precipitated, and resuspended in nuclease-free water. Sequencing libraries were prepared using the CATS Small RNA-seq Kit or D-Plex Small RNA-seq Kit, followed by purification with AMPure XP. Barcoded libraries were pooled and sequenced on Illumina platforms, generating 150 bp paired-end reads.