title: GSM8491739: SI, B6, allogeneic BMT; Mus musculus; RNA-Seq
library_construction_protocol: Intestinal crypt cells were isolated from normal mice or from transplant recipients as follows. In brief, after euthanasia with CO2, either the entire small intestine (SI) or the terminal 10 cm of ileum was collected, opened longitudinally, and washed with PBS. The tissue was then incubated at 4 °C in EDTA (10 mM) for 20 min to dissociate the crypts. To isolate single cells from crypts, the pellet was further incubated in 1× TrypLE express (Gibco, Life Technologies) supplemented with 2 kU/ml DNase1 (Roche). To isolate single cells from intestinal organoids, crypts, organoids were dissociated using 1× TrypLE express at 37°C. After vigorously pipetting through a p200 pipette causing mechanical disruption, the crypt suspension was washed with 10 ml of DMEM/F12 medium containing 10% FBS and 2 kU/ml DNase1 and passaged through a 40 mm cell strainer. Dissociated crypt cells or intestinal organoids were stained with DAPI and then sorted to purify DAPI-negative cells. The single cell RNA sequencing (scRNA-seq) of FACS-sorted cell suspensions was performed on a Chromium instrument (10X genomics) following the user guide manual CG00052 Rev E and Single Cell 3' Reagent Kit (v2). Each sample, containing approximately 5000 cells at a final dilution of 66–70 cells/µl was loaded onto the cartridge according to the manual. The viability of cells was between 83-99%, as confirmed with 0.2% Trypan Blue staining using a Countess II cell counter. The individual transcriptomes of encapsulated cells were barcoded during the reverse transcription (RT) step. The resulting cDNA was purified with DynaBeads. This was followed by amplification with 14 cycles of PCR: 98 ̊C for 180s, 14x (98 ̊C for 15s, 67 ̊C for 20s, 72 ̊C for 60s), and 72 ̊C for 60s. Next, 50 ng of PCR-amplified product was fragmented, A-tailed, purified with 1.2X SPRI beads, ligated to the sequencing adapters, and indexed by PCR: 98 ̊C for 45s; 12x (98 ̊C for 20s, 54 ̊C for 30s, 72 ̊C for 20s), and 72 ̊C for 60s. The indexed DNA libraries were double-size purified (0.6–0.8X) with SPRI beads and sequenced on the Illumina NovaSeq S4 platform (R1 – 26 cycles, i7 – 8 cycles, R2 – 70 cycles or higher). Sequencing depth was 198 million reads per sample on average (31,900 reads per cell). The cell-gene count matrix was constructed using the Sequence Quality Control (SEQC) package48. Viable cells were identified on the basis of library size and complexity, whereas cells with >20% of transcripts derived from mitochondria were excluded from further analysis. After barcode correction and filtering, we recovered 11,427 unique transcripts per cell, with a median read per transcript of 1.83.