GSM7506778: HeLa cell culture, UV crosslinked, RNA binding protein-RNA fraction, replicate two; Homo sapiens; RNA-Seq
Cells were lysed on plate, scraped, and transferred to a 2.0 mL microcentrifuge tube using two 400 µL aliquots of guanidinium thiocyanate (−phenol) buffer. Guanidinium thiocyanate (GT) buffer (4 M GT, 25 mM sodium citrate pH 7.0, 0.5% N-lauryl sarcosine, 5 mM EDTA pH 8.0, and 0.1 M 2-mercaptoethanol) was prepared as described in  [23, 24], with the following stock solutions prepared in DEPC-treated DI water: 5 M guanidinium thiocyanate (00522, Chemimpex), 750 mM sodium citrate pH 7.0 (BDH-9288, VWR; C-0759, Sigma), 10% N-lauryl sarcosine (L9150, Sigma), 0.5 M EDTA pH 8.0 (0105, VWR). Briefly, 3' and 5' adaptors were ligated to 3' and 5' ends of the small RNA fraction, respectively. Then, first strand cDNA was synthesized after hybridization with the reverse transcription primer. The double-stranded cDNA library was generated through PCR enrichment. After purification and size selection, libraries with insertions between 18~40 bp were ready for sequencing on Illumina sequencing with SE50.