title: GSM8505971: NIP1_depl, R42L, replicate 1 [RIBO-seq]; Saccharomyces cerevisiae; OTHER
library_construction_protocol: ~50 A260 unit of cell lysates were supplemented with RNase I and incubated 1 hr at room temperature with gentle rotation. Resulting samples were layered onto 10%-50% sucrose gradient and centrifuged at 39,000 rpm at 4 °C for 2.5 hr using SW-41 Ti rotor. Resulting sucrose gradients were monitor and digested 80S fractions were collected, followed by centrifugal filtration. Sequentially, samples were mixed with release buffer and incubated on ice for 10 min, followed by centrifugation and transferred the flowthrough into a new sterile tube. The flowthroughs were adjusted to 1% SDS, followed by the addition of 1 volume of Acid-Phenol:Chloroform (pH4.5), vortex, incubation at 65°C for 5 min, and incubation on ice for 1 min. Resulting samples were centrifuged at 12,000xg at 4°C for 5 min, and resulting aqueous top was transferred to new sterile 1.5 ml tubes. RNA from the resulting samples were ethanol precipitated by adding NaOAc (pH 5.5) containing 1/100 volume Glycoblue, followed by 2.5 volumes of 100% ethanol and incubation at -20°C overnight. Harvested RNA fragments were dephosphorylated, following adaptor ligation using 3'-adaptor. The Ligated RNAs were reverse transcribed using RT-primer, circularized and PCR-amplified for 12. Following PCR products are purified and subjected to 150bp-double end sequencing using the Illumina Nextseq 1000 system.