Counting absolute number of molecules using unique molecular identifiers
Counting absolute number of molecules using unique molecular identifiers
Drosophila S2 cells were cultured at +23.5 C in a cooling incubator (Binder KB) in Invitrogen SFM medium supplemented with 10% fetal bovine serum, 10% insect medium supplement, Sigma), 2 mM glutamine, and antibiotics. Cells (1 million cells per one well of a 6-well plate seeded the previous day in 2 ml medium) were transfected with dsGFP by  Effectene (Qiagen) transfection reagent according to the manufacturers instructions, except that 1.1 ug of dsRNA, 8.8 ul of enhancer and 5ul of Effectene in EC buffer (125 ul final volume) was used per well. RNA was extracted with Qiagen RNeasy kits according to manufacturers instructions with DNase treatment. Total RNA was fragmented with hydrolysis by 3 min incubation at 70C in 1x RNA fragmentation buffer (Ambion). The reaction was terminated as instructed by manufacturer. The cDNA synthesis was performed according to the SMART protocol18 with addition of adapters for massively-parallel sequencing using an oligo-dT containing adapter (CAAGCAGAAGACGGCATACGAGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTTTVN; Eurofins MWG Operon). For absolute molecule counting, a random ten base DNA sequence label (N) was added to the 5 prime adapter (ACACTCTTTCCCTACACGACGCTCTTCCGATCT(NNN)dU(NNNN)dU(NNN)[GACTT]rGrGrGrG; Integrated DNA technologies). Sequences in square brackets and parentheses represent an index sequence that was used to enable multiplexed sequencing, and a random ten base DNA sequence label, respectively. dU and rG represent deoxyuridine and guanine ribonucleotide, respectively. To denature RNA and anneal the 3 prime adapter, 12 pmol of both adapters were incubated at 72C for 2 min with 3 ml of the unpurified solution containing 50 ng of fragmented total RNA. RNA was then reverse transcribed with SuperScriptIII (Invitrogen; 200 U) in 15 ml volume with the provided buffer, dNTPs (1 mM), DTT (2 mM) and excess MgCl2 (added to 15 mM). The reaction was carried out at 55 C for 1 h and enzyme was inactivated by incubation at 70 C for 15 min. Uracil-specific excision reagent was used to degrade the random label sequence in the template-switch oligonucleotide (5 U of USER per 50 ng of total RNA at 37 C for 30 min; New England Biolabs).  The libraries were amplified using Phusion High-Fidelity DNA polymerase (Finnzymes) from 2 ml of unpurified cDNA reaction mixture with 300 nM Illumina single-read sequencing library primers. PCR was performed according to manufacturers instructions. 20% trehalose was included in the 50 ml reactions, and the following cycle settings were used: denaturation: 1 min at 98 C, followed by 15 to 25 cycles of 10 s at 98 C, 30 s at 64C, and 1 min at 72C. Final extension was 11 min. In the PCR cycle experiment, half of the reaction volume was extracted at cycle 15 and replaced with fresh master mix. PCR products were purified with one volume of Agencourt XP beads (Beckman).  Samples with RNA amounts corresponding to 10, 100 or 1000 Drosophila S2 cells were prepared with ca. 0.07, 0.7 or 7 ng of fragmented RNA, the same GFP treated total RNA that was used in mRNA-seq experiments shown in Figure 3. RNA was synthesized to cDNA using paired-end compatible oligo-dT adapter (12 pmol;CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTTTVN; Integrated DNA technologies) with the labeled 5 prime adapter described above (12 pmol) and SuperScriptIII (Invitrogen; 300 U) in 30 ul volume with the provided buffer, dNTPs (0.8 mM), DTT (2.9 mM) and excess MgCl2 (added to 17 mM). The cDNA was treated with 60 U of Exonuclease I (New England Biolabs) prior to uracil-specific excision reagent. The whole volume of treated cDNA was included in a 100 ul PCR reaction, samples were amplified as described above. Due to library preparation, all sequences begin with NNNTNNNNTNNNGACTTGGGG, where N represents the molecular label (file 25c_repeat1_lane2.fastq  contains also sequences with a distinct barcode from another sample).
Spot Layout
Originally PAIRED end but only Forward reads provided
Experimental Factor: TECHNICAL_REPLICATE
5
Experimental Factor: PCR_CYCLES
30
Experimental Factor: CELL_NUMBER
1000