GSM2565544: PE098-A1-81; Homo sapiens; RNA-Seq
PLATE-Seq: Cells are lysed for PLATE-Seq in Buffer TCL (Qiagen) supplemented with 2-mercaptoethanol in 96-well plates. We transfer 30 uL of cell lysate from each well to a 96-well plate with oligo(dT) grafted to the wells (mRNA TurboCapture Plate, Qiagen) and added 1 uL of 1:100 dilution of ERCC Ex-Fold Spike-Ins (ThermoFisher) to a subset of the sample wells. The plate is then centrifuged at 295xg for 1 minute to eliminate air bubbles followed by incubation for 90 minutes at room temperature with shaking at 200 rpm. Lystate is then removed from the oligo(dT) capture plate and wells are washed three times with Buffer TCW (Qiagen) using the 96 CO-RE Head of a Hamilton Microlab STAR automated liquid handling system. After drying the plate, mRNA is then eluted by adding 30 uL of Elution Buffer (nuclease-free water supplemented with 0.4 U/uL SUPERaseIN (ThermoFisher)), incubation at 65°C for five minutes, and incubation at room temperature for 5 minutes. mRNA samples are then transferred to a new PCR plate (Hard-Shell PCR plate, 96-well, Bio-Rad) using sterile-filter 96-well, 50 uL tips on the Hamilton STAR. To anneal primers for reverse transcription, 3 uL of 100 uM adapter-linked oligo(dT) primers are added to each well along with 10 uL of 5x ProtoScript RT Buffer (New England BioLabs) with the Hamilton STAR CO-RE Head and 1000 uL channel pipetting system. The plate is then heated to 94°C for two minutes and placed immediately on ice for 5 minutes. The remaining components of the reverse transcription master mix including 0.5 mM of each dNTP, 10 mM DTT, 35.8 U of Protoscript II Reverse Transcriptase (New England BioLabs), and water are then added to bring the final reaction volume to 50 uL per well. The plate is then centrifuged at 295xg for 1 minute and incubated at 42°C for 2 hours. At this point, the samples can optionally be frozen at -80°C. To remove excess primer, 1 uL of a four-fold dilution of Exonuclease I (New England BioLabs) is added to each sample. The Exonculease I reaction is incubated at 25°C for one hour. To hydrolyze the RNA, we add 20 uL of a 1:1 mixture of 1M sodium hydroxide and 0.5M EDTA to each well and incubate at 65°C for 15 minutes. The wells are then pooled together into a single tube to which 80 uL of 12 M hydrochloric acid are added to neutralize the sample.  At this point, the pooled sample can optionally be frozen at -80°C. The pooled, neutralized sample is purified and concentrated using a Qiagen MinElute column using a vacuum apparatus according to the manufacturer?s instructions, and pooled cDNA is eluted in 15 uL of nuclease-free water. For second-strand synthesis, 1 uL of 10 mM dNTP mixture and 1 uL of 100 mM adapter-linked random hexamer primers are added to the pooled cDNA sample. The mixture is then heated to 70°C for two minutes and immediately placed on ice for five minutes. Next, 2 uL of NEB Buffer 2 (New England BioLabs) and 1 uL of Klenow Large Fragment DNA Polymerase (New England BioLabs) are added and the reaction is incubated at room temperature for 30 minutes. The reaction is then stopped by addition of EDTA to a final concentration of 50 uM the double-stranded cDNA is purified with two rounds of bead clean-up using AMPure XP beads (Beckman Counter) at a 1:1 bead-to-sample ratio. Finally, the double-stranded cDNA pool is amplified by PCR using Phusion DNA Polymerase (New England BioLabs) with 0.5 uM Illumina RP1 primer and Illumina RPIx primer (where x is a number indicating the Illumina index). The thermocycling protocol begins with 98°C for 30 seconds followed by 10-12 cycles of 98°C for 15 seconds, 62°C for 15 seconds, and 72°C for 60 seconds followed by a seven-minute incubation at 72°C. TruSeq: To compare PLATE-Seq and conventional RNA-Seq, we conducted a small-scale screen in a single 96-well plate.  We treated each well containing cultured BT20 cells with one of seven compounds or DMSO (vehicle) including mitoxantrone, crizotinib, albendazole, idarubicin, aprepitant, gemcitabine, and bortezomib with 12 replicate wells for each condition.  Half of the material in each well was allocated to PLATE-Seq, and 96 libraries were constructed using the automated, pooled procedure described above.  For the other half, we pooled two sets of six wells for each condition to generate 16 samples in total (two replicates for each condition). We then extracted total RNA from each pool using an RNeasy column (Qiagen) and subjected the total RNA to Illumina TruSeq poly(A)+ RNA-Seq library construction according to the manufacturer?s instructions. RNA-Seq using PLATE-Seq or Illumina TruSeq
GEO
GSM2565544
GEO Accession
GSM2565544