GSM9228457: Immunoprecipitated SNP-4 RNP replicate 2 (sperm negative); Caenorhabditis elegans; RIP-Seq
Twenty tubes containing 800 μl of the TEV eluate/TRIzol LS mixture were thawed at room temperature and 160 μl of chloroform was added to each tube and mixed vigorously for 15 seconds. After incubation at room temperature for 10 minutes, the mixture was centrifuged at 12,000 x g for 15 minutes at 4 ºC. Supernatants were collected and applied onto two RNeasy MinElute spin columns using the RNeasy Micro Kit (Qiagen, cat #74004). Finally, about 40 μl of RNA solution was collected from each immunopurification. RNA was quantified using Qubit fluoremetric quantification (Thermo Fisher Scientific, Inc). Total RNA was also extracted from 200 μl of the high-speed supernatant of the lysates using RNeasy Micro Kit (Qiagen, 74004) as a control. A Ribo-Zero rRNA Removal Kit (Illumina, MRZH116) was used to remove ribosomal RNA (rRNA) from the total RNA. Duplicate sequencing libraries were prepared from rRNA-depleted total RNA from the high-speed supernatant of the lysates using the same method so that enrichment over input could be calculated. The number of amplification cycles utilized was determined empirically using 1 μl of the sequencing library in a 10 μl test reaction containing a 1:10,000 dilution of SYBR Green I (Invitrogen), 2x KAPA HiFi HotStart ReadyMix (Roche) and custom barcoding primers. The empirically determined number of amplification cycles was used in a subsequent 50 μl reaction with the remaining eluate in the absence of SYBR Green using Kapa HiFi HotStart Readymix (Roche). Libraries were size-selected on a 2% E-Gel EX agarose gel (Invitrogen)  with visualization using UV-free Dark Reader transilluminator (Claire Chemical Research) and fragments between 250 bp and 400 bp were extracted using a MiniElute Gel Extraction Kit (Qiagen). Libraries were submitted to the University of Minnesota Genomics Center for 2x 36-bp sequencing using the Illumina NextSeq 500 to depth of 37-55 million reads per sample.