GSM5645144: hsts_F4_Crizotinib_0.19_24 HSTSPS1AF04_GTGTACAC; Homo sapiens; RNA-Seq
Cells are lysed for PLATE-Seq in Buffer TCL (Qiagen) supplemented with 2-mercaptoethanol in 96-well plates. We transfer 30 μl 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 μl of 1:100 dilution of ERCC Ex-Fold Spike-Ins (ThermoFisher) to a subset of the sample wells. The plate is then centrifuged at 295 × g for 1 min to eliminate air bubbles followed by incubation for 90 min at room temperature with shaking at 200 rpm. Lysate 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 μl of elution buffer (nuclease-free water supplemented with 0.4 U μl−1 SUPERaseIN (ThermoFisher)), incubation at 65 °C for 5 min, and incubation at room temperature for 5 min. mRNA samples are then transferred to a new PCR plate (Hard-Shell PCR plate, 96-well, Bio-Rad) using sterile-filter 96-well, 50 μl tips on the Hamilton STAR. To anneal primers for reverse transcription, 3 μl of 100 μM adapter-linked oligo(dT) primers are added to each well along with 10 μl of 5× ProtoScript RT Buffer (New England BioLabs) with the Hamilton STAR CO-RE Head and 1000 μl channel pipetting system (see Supplementary Table 2 for “PLATEseq_oligodTBC” sequences). The plate is then heated to 94 °C for 2 min and placed immediately on ice for 5 min. 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 μl per well. The plate is then centrifuged at 295 × g for 1 min and incubated at 42 °C for 2 h. At this point, the samples can optionally be frozen at −80 °C. To remove excess primer, 1 μl of a fourfold dilution of Exonuclease I (New England BioLabs) is added to each sample. The Exonculease I reaction is incubated at 25 °C for 1 h. To hydrolyze the RNA, we add 20 μl of a 1:1 mixture of 1 M sodium hydroxide and 0.5 M EDTA to each well and incubate at 65 °C for 15 min. The wells are then pooled together into a single tube to which 80 μl 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 μl of nuclease-free water. For second-strand synthesis, 1 μl of 10 mM dNTP mixture and 1 μl of 100 mM adapter-linked random hexamer primers are added to the pooled cDNA sample. The mixture is then heated to 70 °C for 2 min and immediately placed on ice for 5 min. Next, 2 μl of NEB Buffer 2 (New England BioLabs) and 1 μl of Klenow large fragment DNA polymerase (New England BioLabs) are added and the reaction is incubated at room temperature for 30 min. The reaction is then stopped by addition of EDTA to a final concentration of 50 μM 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) RNA libraries were prepared for sequencing using standard Illumina protocols