title: GSM8250372: SeqScope_2nd colon; Mus musculus; OTHER
library_construction_protocol: The Seq-Scope procedure was performed on the injured colon sample as previously described with minor modifications involving the use of the Illumina HiSeq 2500 flow cells and chemical strategies to increase RNA sequence capture. The experimental procedures were performed as we previously described, but it was adapted to HISEQ2500 flow cell system so that Seq-Scope has wider and continuous imaging area (1.5 mm × 60 mm in HISEQ2500, compared to 1 mm-diameter circles in MISEQ). A single imaging area was used for many samples. In brief, HDMI32-Dral [Seed HDMI-oligo, containing the PCR/read adaptor sequences, the restriction enzyme-cleavable RNA-capture domain (olido-dT), the high-definition map coordinate identifier (HDMI), a spatial barcode composed of a 32 random nucleotide sequence] was amplified on a flow cell surface to generate HDMI-oligo cluster. The HDMI sequence and coordinates of each cluster were determined using the HISEQ2500 platform. Thumbnail images of clusters were visualized using Illumina Sequencing Analysis Viewer to inspect the cluster morphology and density. Next, the HISEQ2500 flow cell was processed to expose the oligo-dT and RNA-capture domain and generate an HDMI-encoded RNA-capturing array (HDMI-array). Subsequently, the HISEQ2500 flow cell was disassembled to expose the HDMI-array to the outside and used for tissue attachment. The OCT-mounted fresh frozen aortic tissue was sectioned 10 μm thickness in a cryostat (Leica CM3050S, -20°C) and the aortic sections were maneuvered onto the HDMI-array slide. After fixing in 4% formaldehyde for 10 min and incubating in isopropanol for 2 min, the tissue was stained with hematoxylin (Agilent, S3309) for 5 min, treated with bluing buffer (Agilent, CS702) for 2 min, and incubated with buffered eosin [1:9 = eosin (HT110216, Sigma): 0.45M Tris-Acetic buffer (pH 6.0)]. After mounting in 85% glycerol, the tissues were imaged under a digital imager (Keyence, BZ-X800). Total RNAs released from the fixed tissues were performed as previously described. Briefly, the fixed tissues attached on the HDMI-array slide were treated with 0.2 U/μl collagenase I (Thermo Fisher, 17018-029) at 37 °C for 20 min, and then incubated with 1mg/ml pepsin (Sigma, P7000) in 0.1M HCl at 37 °C for 10 min. The RNAs released from the tissue sections were captured by the HDMI-array and used as a template to generate cDNA footprints on the HDMI-barcoded RNA capturing molecule by reverse transcription. After overnight reverse transcription reaction at 37 °C, the tissues were incubated in exonuclease I cocktail [1U Exo I enzyme (M2903, NEB) in 1x Exo I buffer] and subsequently digested in 1x tissue digestion buffer [100 mM Tris pH8.0, 100mM NaCl, 2% SDS, 5mM EDTA, 16 U/ml Proteinase K (P8107S, NEB)]. After tissue digestion, the secondary strand, a chimeric molecule of HDMI and cDNA sequences, is synthesized on the cDNA footprint using an adaptor-tagged random primer (Truseq Read 2-conjugated Random Primer version 2 with TCA GAC GTG TGC TCT TCC GAT CTN NNN NNN NB sequence). After removing all DNAs that did not bind to the HDMI-array, the secondary strand product was eluted using NaOH, and after neutralization, purified using AMPure XP purification kit (A63881, Beckman Coulter). The secondary strand product was used as a template to construct library using Kapa HiFi Hotstart Readymix (KK2602, KAPA Biosystems) through two rounds of library PCR. After purification using Zymoclean Gel DNA recovery Kit (D4001, Zymo Research) and AMPure XP, the pooled libraries were subjected to paired-end (100-150bp) sequencing in the Illumina platform at Psomagen lnc.