GSM3415780: Lu_5; Homo sapiens; RNA-Seq
For solid tissue biosamples, RNA extraction was performed immediately before preparation of sequencing libraries using QIAGEN RNeasy Kit (Qiagen) for tissues in RNAlater and using the RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Invitrogen) following the manufacturer 's protocol. Then RNA Integrity Number (RIN) was measured using Agilent 2100 bio-Analyzer. Agilent RNA 6000 Nano or Qubit RNA Assay Kits were used to measure RNA concentration. The Kit for further depletion of ribosomal RNA was selected based on total amount of RNA in every sample: (i) when total amounts of RNA were less than 25 ng, Clontech Smarter stranded total RNA kit-pico (Mammalian only) was used; (ii) when total amount of RNA exceeded 25ng, KAPA RNA Hyper with RiboErase (KAPA Biosystem) kits were used. For library preparations, we used Ovation® Universal RNA-Seq System 1-96 and KAPA HyperPrep Kit according to the manufacturer's recommendations. Different adaptors were used for multiplexing samples in one sequencing run. Library concentrations and quality were measured using Qubit ds DNA HS Assay kit (Life Technologies) and Agilent Tapestation (Agilent). RNA sequencing was performed using Illumina HiSeq 3000 equipment for single-end sequencing, 50 bp read length, for approx. 30 million raw reads per each sample. Data quality check was done on Illumina SAV. De-multiplexing was performed with Illumina Bcl2fastq2 v 2.17 program.