title: GSM3074504: Hela_cell_RNC_seq; Homo sapiens; RNA-Seq
library_construction_protocol: Hela and MHCCLM3 and MHCC97H cells were washed by pre-chilled phosphate buffered saline twice and addition of 2 ml cell lysis buffer [1% Triton X-100 in ribosome buffer (RB buffer) [20 mM HEPES-KOH (pH 7.4), 15 mM MgCl2, 200mM KCl, 100 mg/ml cycloheximide and 2mM dithiothreitol]. After 30-min ice-bath, cell lysates were scraped and transferred to pre-chilled 1.5 ml tubes. Cell debris was removed by centrifuging at 16 200 g for 10 min at 4C. Supernatants were transferred on the surface of 20 ml of sucrose buffer (30% sucrose in RB buffer). RNCs were pelleted after ultra-centrifugation at 185000 g for 5h at 4C. Then RNC-RNA were isolated by using TRIzol RNA extraction reagent (Ambion, Austin, TX), following the manufacturer's instructions. The Hela RNC-mRNA libraries were prepared according to BGI-MGIEasy's instructions accompanying the MGIEasy mRNA Library Prep Kit V2 (Part# 85-05536-01). The polyA+ mRNA was selected using NEB Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). The mRNA libraries were constructed using the MGIEasy mRNA Library Prep Kit V2 for MGI (BGISEQ, Shen Zhen, China) following the manufacturer's protocol. The MHCC97H RNC-mRNA  libraries were prepared according to VAHTS mRNA-seq V3 Library Prep Kit (Part# NR611-01/02). The polyA+ mRNA was selected using NEB Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). The mRNA libraries were constructed using the VAHTS mRNA-seq V3 Library Prep Kit for Illumina® (Vazyme, Nanjing, China) following the manufacturer's protocol. RNA-Seq (Hela cell RNC-mRNA sequencing were performed on a BGISEQ-500 sequencer for single-end 50, and the MHCC97H RNC-mRNA sequencing were performed on an Illumina HiSeq X Ten sequencer for paired-end 150.
attributes.tag: GEO Accession
attributes.value: GSM3074504