GSM6333420: THP-1 cells, Riboseq/LPS, 3h, rep3; Homo sapiens; OTHER
The lysate was homogenized by immediate repeated pipetting and multiple passes through a syringe fitted with a 21G needle, allowing for dissociation of cell clumps and facilitating quick and equal lysis of the cells. Samples were next centrifuged at 20,000g for 10 minutes at 4°C to pellet cell debris. Per sample, 200 or 400µL of lysate were digested with RNase 1 (Biozym), purified with Microspin Sephacryl S-400 HR columns (Sigma-Aldrich) and 1µg footprints were used for removal of the ribosomal RNA according to the Ribo-Zero Gold rRNA Removal Kit (h/m/r) (Illumina). The footprints were purified by excision from a Novex 15% TBE Urea PAGE gel (Fisher Scientific) followed by a treatment of the 3´ends with T4 PNK (Biozym) to allow ligation to a pre-adenylated linker. The RNA was then reverse transcripted with RNase H Reverse Transcriptase (Biozym)  and the cDNA purified via a Novex 10% TBE Urea PAGE gel (Fisher Scientific). The fragments were circularized using Circligase I (Biozym) followed by a PCR amplification and size selection using a Novex 8% TBE PAGE gel (Fisher Scientific). For all samples, ribosome profiling library size distributions were checked on the Bioanalyzer 2100 using a high sensitivity DNA assay (Agilent), multiplexed and sequenced on a NovaSeq 6000 producing single end 1x51nt reads. Ribo-seq libraries were sequenced to an average depth of 80M (min. 64M, max. 91M) raw reads. Ribosome profiling library was constructed according to previously described work (McGlincy NJ, 2017; van Heesch S, 2019; Gaertner B, 2020 ). See also the extraction protocol.