design: Illumina Paired-end Sequencing of Human Cell Line HCC1954 Transcriptome
library_construction_protocol: Total RNA was isolated from the cell pellets using the RNeasy Mini Kit (Qiagen, Valencia, CA). Total RNA was treated with DNase I (New England Biolabs, Ipswich, MA) and purified with Qiagen RNeasy columns (Qiagen Valencia, CA). DNA-free RNA yield and purity were initially assessed by spectrophotometry. PolyA+ RNA was prepared from 500 µg of total RNA with oligo(dT) beads using the Oligotex mRNA Mini Kit (Qiagen Valencia, CA). First-strand cDNA was prepared from 1 µg of poly(A)+ RNA with 200 pmol oligo random primers by using 300 units of Superscript II reverse transcriptase (Invitrogen , Carlsbad, CA). Second-strand synthesis was performed in 20 µl at 16oC for 2 h after addition of 10 units of E. coli DNA ligase, 40 units of E. coli DNA polymerase, and 2 units of RNase H (all from Invitrogen, Carlsbad, CA). T4 DNA polymerase (5 units) was added and incubated for 5 min at 16oC. Double-strand cDNA was purified by phenol-chloroform extraction and precipitation of the aqueous phase in 1/10 volume 3 M sodium acetate and 100% ethanol.