GSM6339317: 375KO_set2; Mus musculus; RNA-Seq
Mouse jejunal crypts from male B6 albino wildtype and miR-375 knockout mice were isolated and resuspended in an ice-cold PBS containing 0.04% (w/v) bovine serum albumin and pelleted at 1000 x g at 4°C for 5 min. The crypts were subsequently digested with 0.3 U/mL Dispase I (MilliporeSigma Burlington, MA; D4818) in HBSS at 37°C for 12 min with gentle agitation. The dispase I activity was stopped by adding fetal bovine serum to a final concentration of 10% (v/v) and DNaseI to a final concentration of 50 μg/mL. The sample was filtered through 40 μm strainer, pelleted at 500 x g at 4°C for 5 min, washed with cold HBSS, filtered again, and then resuspended in an ice-cold PBS containing 0.04% (w/v) bovine serum albumin. Prior to submission, single cell suspensions were triturated by pipetting and evaluated for total viable cell number by using trypan blue staining with a TC20 automated cell counter (Bio-Rad Laboratories, Richmond, CA). Single-cell RNA sequencing of these samples was performed at the Cornell University Biotechnology Resource Center. Libraries were prepared using the 10X Genomics Chromium preparation kit (3' v2 chemistry)(10X Genomics, Pleasanton, CA). Briefly, single cell suspension were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriate sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.