GSM8988840: Parental-Ribo-Seq- Replicate 3; Homo sapiens; RNA-Seq
Flag/HA control, ZC3H7A-OE, or ZC3H7B-OE HEK293 cells (3 independent replicates each) were lysed in hypotonic buffer (5 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2, 1.5 mM KCl, 1x EDTA-free protease inhibitor cocktail, 2 mM DTT, 200 U/ml RiboLock RNase Inhibitor, 0.5% (v/w) Triton X-100, and 0.5% (v/w) Sodium Deoxycholate), to isolate the polysomes. 500 µg of the lysates were subjected to ribosome footprinting by RNase I. Flash frozen ZC3H7A/B-DD and Parental cell pellets were lysed in ice-cold polysome lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100) supplemented with cycloheximide (100 µg/mL) (Sigma-Aldrich, Cat. # 01810). The Ribo-Seq libraries for Flag/HA-control, Flag/HA-ZC3H7A, and Flag/HA-ZC3H7B samples were generated via the NEXTFLEX® Small RNA-Seq Kit v3. The RNA-Seq libraries for Flag/HA-control, Flag/HA-ZC3H7A, and Flag/HA-ZC3H7B samples were generated via the Illumina TruSeq RNA Sample Preparation Kit v2. The Ribo-Seq libraries for the ZC3H7A/B-DD and Parental cells were generated using Illumina compatible protocols by EIRNA Bio.