GSM5736169: smRNA-seq_HCT15_1; Homo sapiens; miRNA-Seq
Total RNA was isolated from samples using TRIzol Reagent (Invitrogen, USA) and purified using RNeasy Mini Kit (QIAGEN, Germany). Agilent Bioanalyzer 2100 was used to assess RNA integrity. The mRNA and lncRNA‐seq library preparation from 3 μg of total RNA was performed with NEBNext UltraTM Directional RNA Library Prep Kit for Illumina (NEB, USA) according to the manufacturer's instructions. PE150 sequencing was performed on the Illumina HiSeq X Ten platform (Novogene Biotech, China). A total amount of 3 μg total RNA per sample was used as input material for the small RNA library. NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, USA) was used to generate the sequencing libraries following the manufacturer's instructions. library quality was assessed on the Agilent Bioanalyzer 2100 system and then sequenced on an Illumina Hiseq 2500 platform (Novogene Biotech, China). 50bp single-end reads were generated for each sample. The ATAC-seq libraries were prepared using a total of 50,000 fresh cells were washed twice with 100 μl of cold PBS (KEL Biotech, China) and resuspended in 100 μl of lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 0.1% IGEPAL CA-630 (Sigma-Aldrich) and 3 mM MgCl2] for 10 min on ice to prepare the nuclei. Immediately after lysis, the suspension of nuclei was spun at 500 g at 4°C for 5 min to remove the supernatant. Nuclei were then incubated with the 50 μl of Tn5 transposition reaction mix (DIATRE Biotech, China) at 37 °C for 30 min. The stop buffer was then added directly into the reaction to end the tagmentation. DNA was purified using the QIAquick PCR Purification Kit (QIAGEN, Germany). PCR was performed to amplify the library for 12 cycles. Libraries were purified with Agencourt Ampure XP beads (Beckman, USA) to remove contaminating primer dimmers.