Second strand buffer

Next, 1,100 nl of second-strand synthesis mix (1.14× Second-Strand Buffer (Invitrogen), 0.23 mM (each) dNTP mix (Promega), 0.35 U of E. coli DNA Polymerase I (Invitrogen) and 20 mU of RNaseH (Invitrogen)) was added to each well. Plates were sealed and spun down at 2,000 r.c.f. for 2 minutes. Second-strand synthesis was carried out at 16 °C for 2 hours, followed by 85 °C for 20 minutes. Plates were snap-chilled, spun down at 2,000 r.c.f. for 1 minute and stored on ice before pooling. The protocol for pooling and in vitro transcription (IVT) was the same as SORT-seq^{27 (link)}.

The eluted cDNA:mRNA hybrids (15 μl) were combined with a second-strand mix [2 μl of 5 × First-Strand Buffer (Invitrogen), 2.31 μl of Second Strand Buffer (Invitrogen), 0.23 μl of 10 mM dNTPs (NEB), 0.08 μl of 10 U/μl E. coli DNA ligase (Invitrogen), 0.3 μl of 10 U/μl E. coli DNA polymerase (Invitrogen) and 0.08 μl of 2 U/μl E. coli RNaseH (Invitrogen)] and incubated for 2 h at 16 °C. The double-stranded cDNAs were purified with AMpure XP beads (Beckman Coulter) and eluted with H₂O.

Permeabilization mixture was prepared as described in the optimization microarray experiment. cDNA mixture for library preparation differed from optimization mixture in the way that it did not contain fluorescently labeled Cy3-dCTP. Mixture contained: 1× First Strand Buffer (Invitrogen), 5 mM DTT (Invitrogen), 0.5 mM of each dNTP (Fisher Scientific), 0.19 μg μl^–1 BSA, 50 ng μl^–1 actinomycin D (Sigma-Aldrich), 1% DMSO (Sigma-Aldrich), 20 U μl^–1 Superscript III (Invitrogen) and 2 U μl^–1 RNaseOUT (Invitrogen) and cDNA was synthesized over night at 42°C. After the cDNA was synthesized, cells were removed with proteinase K in the PKD buffer (1:4 ratio) and subsequently washed as described in an optimization microarray experiment.
Probe release. Release of probes with mRNA–cDNA hybrids from the array surface was performed by adding 70 μl of release mix to the array chambers. Release mix was composed of 1× Second Strand Buffer (Invitrogen), 8.75 μM of each dNTP, 0.20 μg μl–1 BSA, and 0.1 U μl–1 USER enzyme (NEB). Incubation was performed at 37°C for 2 hours with interval shaking and 300 r.p.m. Release mixture was collected and transferred to a 96 well plate and placed at -20°C until further processing with an automatic robotic station.

First-strand cDNA synthesis was performed with 195 ng total RNA using anchored oligo-dT and SuperScript III First-Strand Synthesis SuperMix (Thermo Fisher Scientific). Second strand cDNA synthesis was performed using RNase H, DNA polymerase I, and Second Strand Buffer (Thermo Fisher Scientific). Double-stranded cDNA was purified using DNA Clean & Concentrator-5 (Zymo, Irvine, CA). Library preparation was performed using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA). Libraries were cleaned using Agencourt AMPure XP beads according to manufacturer's instructions (Beckman Coulter). Libraries were evaluated by the High Sensitivity DNA Kit on the 2100 Bioanalyzer. They were then multiplexed and sequenced on an Illumina HiSeq with 50 bp paired-end reads. Reads were aligned to NCBI build 37.2 using Tophat (v2.1.0).²² (link) Cuffquant and Cuffnorm (Cufflinks v2.2.1) were used to quantify expression levels and calculate normalized fragments per kilobase of transcript per million mapped reads (FPKM) values.²³ (link)

For tissue removal the two-step protocol was used^{70 (link)}, in brief beta-Mercaptoethanol (Calbiochem, 444203) was diluted in RNeasy lysis buffer (Qiagen, 79216) and the slide was incubated for 1 h at 56 °C. The wells were washed with 0.1× SSC followed by incubation with proteinase K (Qiagen, 19131), diluted in proteinase K digestion buffer (Qiagen, 1034963), for 1 h at 56 °C. The wells were then washed in 2× SSC + 0.1% SDS followed by 0.2× SSC and lastly with 0.1× SSC and dried. The released mix consisting of second-strand buffer (Thermo Fisher, 10812014), dNTPs (Thermo Fisher, R0191), BSA (Bionordika, B9000S), and USER enzyme (Bionordika, M5505L) was added to each well and incubated for 2 h at 37 °C. After probe release, the 1007 spatial spots containing non-released DNA oligonucleotide fragments were detected by hybridization of fluorescently labeled probes (Supplementary Table 3) and imaging, in order to obtain Cy3-images for image alignment and spot detection, as described in the protocol ^{24 (link),70 (link)}.