EK 293T cells were cultured in parallel in 2*150 cm3 flasks with DMEM (penicillin-streptomycin) (Gibco) supplemented with 10% Fetal Calf Serum (FCS) (Biochrom) and Glutamax (1x) (Gibco). At passage 20 (confluent state), cells were trypsinized, washed off in PBS, pelleted and resuspended in 2 ml lysis solution RLT (Qiagen). B cells were cultured parallely in 2*150 cm3 flasks with RPMI 1640 (penicillin-streptomycin) (Gibco) supplemented with 10% FCS (Biochrom) and Glutamax (1x) (Gibco). At passage 20 (confluent state), cells were trypsinized, washed off in PBS, pelleted, and resuspended in 2 ml lysis solution RLT (Qiagen).
Extracted molecule
polyA RNA
Extraction protocol
Total RNA was then extracted from ~ 20 x 106 cells per sample (2*HEK and 2*B cells samples) using RNeasy Midi extraction kit (Qiagen) by following the manufacturer’s instructions. DNA was removed using the “on column digestion” protocol of the RNeasy Midi extraction kit (Qiagen). Total RNA quality was assessed by spectrophotometry (Nanodrop) and gel electrophoresis (1% agarose). Then, mRNA was extracted from 120 µg of each total RNA sample, using Dynabeads mRNA purification kit (Invitrogen) and following the manufacturer’s instructions. The mRNA was eluted in 10,5 µl 10 mM Tris-HCl. First strand cDNA was directly generated from the eluted mRNA using random hexamers (Invitrogen) and superscript RT kit (Invitrogen), following the manufacturer’s instructions. The final volume was 20 µl. The second strand cDNA synthesis was generated immediately after the first strand synthesis. Briefly, 1x Second strand buffer (Invitrogen), 200 nM final dNTPs (Invitrogen), 20 Units of E. coli DNA ligase (Invitrogen), 40 Units of E. coli polymerase (Invitrogen), and 4 Units of E. coli RNase H (Invitrogen) were added to the first strand cDNA and incubated for 2 hours at 16°C. Double stranded cDNA was then purified using QIA quick PCR purification kit, following the manufacturer’s instructions. The generated cDNA was quantified by UV spectrophotometry (Nanodrop). For the digital expression libraries, about 250 ng of the double-stranded cDNA of each sample was fragmented by sonication on the UTR200 (Hielscher Ultrasonics GmbH, Germany) under following conditions: 1 hour, 50% pulse, 100% power, and continuous cooling by 0°C water flow-through. In brief the library preparation consisted of the following steps: end-repair, A-tailing, ligation of adapter primers, size selection and pre-amplification. For all samples, the libraries were prepared using the DNA sample kit (#FC-102-1002, Illumina), following the manufacturer’s instructions, but with the following modifications: five times reduced amount of all enzymes were used and adapters were ligated to the DNA fragments using 2 µl of ‘Adapter oligo mix’ in a total reaction volume of 25 µl. For the digital expression library, 250 ng of the sheared double stranded cDNA (see above) were used and 170-220bp fragments were selected at the gel size fractionation step.
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
other
Instrument model
Illumina Genome Analyzer
Description
raw sequence 070612_4
Data processing
Reads were aligned to the human genome (hg18, NCBI build 36.1) using Eland software (Gerald module v.1.27, Illumina). The mapping criteria imposed by Eland are the followings: matches should be collinear to the genome allowing up to two mismatches, but no indels. In these conditions, 50% of the reads obtained here matched to unique locations of the human genome, whereas 16-18% of the reads mapped to more than one genomic position. Reads that were found in unique positions in the human genome were further mapped to exons of protein-coding genes retrieved from two independent databases: ENSEMBL v.46 and Eldorado (release 05/2007). The number of reads that were fully included in exons (which we refer as “number of hits”) was counted. For genes encoding multiple transcripts, the number of hits per gene was determined as the sum of all hits in all possible exons.
